Amyloid-beta binding proteins

ABSTRACT

The present invention relates to amyloid-beta (Aβ) binding proteins. Antibodies of the invention have high affinity to Aβ(20-42) globulomer or any Aβ form that comprises the globulomer epitope. Method of making and method of using the antibodies of the invention are also provided.

This is a divisional of U.S. patent application Ser. No. 13/085,891,filed on Apr. 13, 2011, now U.S. Pat. No. 8,987,419, which claimspriority to U.S. Patent Application No. 61/446,624, filed on Feb. 25,2011, U.S. Patent Application No. 61/373,825, filed on Aug. 14, 2010,and U.S. Patent Application No 61/324,386, filed on Apr. 15, 2010, theentire contents of all of which are fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to amyloid-beta (Aβ) binding proteins,nucleic acids encoding said proteins, methods of producing saidproteins, compositions comprising said proteins and the use of saidproteins in diagnosis, treatment and prevention of conditions such asamyloidoses, e.g., Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a neurodegenerative disorder characterizedby a progressive loss of cognitive abilities and by characteristicneuropathological features comprising deposits of amyloid beta (Aβ)peptide, neurofibrillary tangles and neuronal loss in several regions ofthe brain (Hardy and Selkoe, Science 297: 353, 2002; Mattson, Nature431: 7004, 2004. Cerebral amyloid deposits and cognitive impairmentsvery similar to those observed in Alzheimer's disease are also hallmarksof Down syndrome (trisomy 21), which occurs at a frequency of about 1 in800 births.

The Aβ peptide arises from the amyloid precursor protein (APP) byproteolytic processing. This processing is effected by the cooperativeactivity of several proteases named α-, β- and γ-secretase and leads toa number of specific fragments of differing length. The amyloid depositsconsist mostly of peptides with a length of 40 or 42 amino acids (Aβ40,Aβ42). This also includes, in addition to human variants, isoforms ofthe amyloid β(1-42) protein present in organisms other than humans, inparticular, other mammals, especially rats. This protein, which tends topolymerize in an aqueous environment, may be present in very differentmolecular forms. A simple correlation of the deposition of insolubleprotein with the occurrence or progression of dementia disorders suchas, for example, Alzheimer's disease, has proved to be unconvincing(Terry et al., Ann. Neurol. 30: 572-580, 1991; Dickson et al.,Neurobiol. Aging 16: 285-298, 1995). In contrast, the loss of synapsesand cognitive perception seems to correlate better with soluble forms ofAβ(1-42) (Lue et al., Am. J. Pathol. 155: 853-862, 1999; McLean et al.,Ann. Neurol. 46: 860-866, 1999).

None of the polyclonal and monoclonal antibodies which have been raisedin the past against monomeric Aβ(1-42) have proven to produce thedesired therapeutic effect without also causing serious side effects inanimals and/or humans. For example, passive immunization results frompreclinical studies in very old APP23 mice which received a N-terminaldirected anti-Aβ(1-42) antibody once weekly for 5 months indicatetherapeutically relevant side effects. In particular, these mice showedan increase in number and severity of microhemorrhages compared tosaline-treated mice (Pfeifer et al., Science 298: 1379, 2002). A similarincrease in hemorrhages was also described for very old (>24 months)Tg2576 and PDAPP mice (Wilcock et al., J Neuroscience 23: 3745-51, 2003;Racke et al., J Neuroscience 25: 629-636, 2005). In both strains,injection of anti-Aβ(1-42) resulted in a significant increase ofmicrohemorrhages.

WO 2004/067561 refers to globular oligomers (“globulomers”) of Aβ(1-42)peptide and a process for preparing them. WO 2006/094724 relates tonon-diffusible globular Aβ(X-38 . . . 43) oligomers wherein X isselected from the group consisting of numbers 1 . . . 24. WO 2004/067561and WO 2006/094724 further describes that limited proteolysis of theglobulomers yields truncated versions of said globulomers such asAβ(20-42) or Aβ(12-42) globulomers. WO 2007/064917 describes thecloning, expression and isolation of recombinant forms of amyloid βpeptide (referred to hereafter as N-Met Aβ(1-42)) and globulomeric formsthereof. The data suggest the existence of an amyloid fibril independentpathway of Aβ folding and assembly into Aβ oligomers which display oneor more unique epitopes (hereinafter referred to as the globulomerepitopes). Since globulomer epitopes were detected in the brain of ADpatients and APP transgenic mice and the globulomer specifically bindsto neurons and blocks LTP, the globulomer represents a pathologicallyrelevant Aβ conformer. It has been found that soluble Aβ globulomerexert its detrimental effects essentially by interaction with the P/Qtype presynaptic calcium channel, and that inhibitors of thisinteraction are therefore useful for treatment of amyloidoses such asAlzheimer's disease (WO 2008/104385).

Antibodies which selectively bind to such globulomeric forms of Aβ havebeen described in WO 2007/064972, WO 2007/062852, WO 2008067464, WO2008/150946 and WO 2008/150949. For instance, several monoclonalantibodies known from WO 2007/062852 and WO 2008/150949 specificallyrecognize Aβ(20-42) globulomer.

There exists a tremendous, unmet therapeutic need for the development ofbiologics such as Aβ binding proteins that prevent or slow down theprogression of the disease without inducing negative and potentiallylethal effects on the human body. Such a need is particularly evident inview of the increasing longevity of the general population and, withthis increase, an associated rise in the number of patients annuallydiagnosed with Alzheimer's disease or related disorders. Further, suchAβ binding proteins will allow for proper diagnosis of Alzheimer'sdisease in a patient experiencing symptoms thereof, a diagnosis whichcan only be confirmed upon autopsy at the present time. Additionally,the Aβ binding proteins will allow for the elucidation of the biologicalproperties of the proteins and other biological factors responsible forthis debilitating disease.

SUMMARY OF THE INVENTION

The present invention provides a novel family of Aβ binding proteins (orsimply “binding proteins”), CDR grafted antibodies, humanizedantibodies, and fragments thereof, capable of binding to soluble Aβglobulomers, for example, Aβ(20-42) globulomer as described herein. Itis noted that the binding proteins of the present invention may also bereactive with (i.e. bind to) Aβ forms other than the Aβ globulomersdescribed herein, such Aβ forms may be present in the brain of a patienthaving an amyloidosis such as Alzheimer's disease. These Aβ forms may ormay not be oligomeric or globulomeric. The Aβ forms to which the bindingproteins of the present invention bind include any Aβ form thatcomprises the globulomer epitope with which the murine/mouse monoclonalantibody m4D10 is reactive (hereinafter referred to as “m4D10”). m4D10and its properties are described in WO 2007/062852, which isincorporated herein by reference. Such Aβ forms are hereinafter referredto as “targeted Aβ forms”. Further, the present invention also providesa therapeutic means with which to inhibit the activity of said targetedAβ forms and provides compositions and methods for treating diseasesassociated with said targeted Aβ forms, particularly amyloidosis such asAlzheimer's disease.

In one aspect, the invention provides a binding protein comprising: afirst amino acid sequence which is at least 90% identical to

SEQ ID NO:2:

EVQLVESGGGLX¹²QPGGSLRLSCAX²⁴SGFTX²⁹SSYGVHWVRQAPGKGLEWX⁴⁸X⁴⁹VIWRGGRIDYNAAFMSRX⁶⁷TISX⁷¹DNSKX⁷⁶TX⁷⁸YLQMNSLRAEDTAVYYCARNSDVWGQGTTVTVSS,wherein X¹² is I or V, X²⁴ is A or V, X²⁹ is V or L, X⁴⁸ is V or L, X⁴⁹is S or G, X⁶⁷ is F or L, X⁷¹ is R or K, X⁷⁶ is N or S, and X⁷⁸ is L orV; orSEQ ID NO:3:

X¹VQLQESGPGLVKPSETLSLTCTVSGX²⁷SX²⁹SSYGVHWX³⁷RQPPGKGLEWX⁴⁸GVIWRGGRIDYNAAFMSRX⁶⁷TISX⁷¹DTSKX⁷⁶QX⁷⁸SLKLSSVTAADTAVYYCARNSDVWGQGTTVTVSS,wherein X¹ is Q or E, X²⁷ is G or F, X²⁹ is I or L, X³⁷ is I or V, X⁴⁸is I or L, X⁶⁷ is V or L, X⁷¹ is V or K, X⁷⁶ is N or S, and X⁷⁸ is F orV;and a second amino acid sequence which is at least 90% identical toSEQ ID NO:1:

DVVMTQX⁷PLSLPVTX¹⁵GQPASISCKSSQSLLDIDGKTYLNWX⁴¹X⁴²QX⁴⁴PGQSPX⁵⁰RLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR,wherein X⁷ is S or T, X¹⁵ is L or P, X⁴¹ is F or L, X⁴² is Q or L, X⁴⁴is R or K, and X⁵⁰ is R or Q.

In a further aspect of the invention, the binding protein describedabove comprises a first amino acid sequence which is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, andSEQ ID NO:11. In still a further aspect of the invention, the bindingprotein described above comprises a first amino acid sequence selectedfrom the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

In another aspect of the invention, the binding protein described abovecomprises a second amino acid sequence which is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16. In still another aspect of theinvention, the binding protein described above comprises a second aminoacid sequence selected from the group consisting of SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

In one aspect of the invention, the binding protein described abovecomprises a first amino acid sequence which is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ IDNO:11; and a second amino acid sequence which is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16. In a further aspect of theinvention, the binding protein described above comprises a first aminoacid sequence selected from the group consisting of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and SEQ ID NO:11; and a second amino acid sequence selected from thegroup consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16.

In a particular aspect of the invention, the binding protein describedabove comprises a first amino acid sequence which is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence set forth in SEQ ID NO:6; and a second amino acid sequencewhich is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth in SEQ ID NO:14. In afurther particular aspect of the invention, the binding proteindescribed above comprises a first amino acid sequence set forth in SEQID NO:6; and a second amino acid sequence set forth in SEQ ID NO:14.

In a particular aspect of the invention, the binding protein describedabove comprises a first amino acid sequence which is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence set forth in SEQ ID NO:10; and a second amino acid sequencewhich is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to an amino acid sequence set forth in SEQ ID NO:14. In afurther particular aspect of the invention, the binding proteindescribed above comprises a first amino acid sequence set forth in SEQID NO:10; and a second amino acid sequence set forth in SEQ ID NO:14.

In one aspect, the binding protein described herein is an antibody. Thisantibody may be, for example, an immunoglobulin molecule, a disulfidelinked Fv, a monoclonal antibody (mab), a single chain Fv (scFv), achimeric antibody, a single domain antibody, a CDR-grafted antibody, adiabody, a humanized antibody, a multispecific antibody, a Fab, a dualspecific antibody, a dual variable domain (DVD) binding molecule, aFab′, a bispecific antibody, a F(ab′)₂, or a Fv.

When the binding protein described herein is an antibody, it comprisesat least one variable heavy chain that corresponds to the first aminoacid sequence as defined above, and at least one variable light chainthat corresponds to the second amino acid sequence as defined above. Forexample, an antibody of the invention comprises (i) at least onevariable heavy chain comprising an amino acid sequence which is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and (ii) at least onevariable light chain comprising an amino acid sequence which is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ IDNO:16. In a particular aspect of the invention, the antibody of theinvention comprises (i) at least one variable heavy chain comprising anamino acid sequence which is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to an amino acid sequence set forth inSEQ ID NO:6 or SEQ ID NO:10, and (ii) at least one variable light chaincomprising an amino acid sequence which is at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99° A or 100% identical to an amino acidsequence set forth in SEQ ID NO:14.

The binding protein described herein may further (in addition to thefirst and second amino acid sequence) comprise another moiety which maybe another amino acid sequence or other chemical moiety. For instance,an antibody of the present invention may comprise a heavy chainimmunoglobulin constant domain. Said heavy chain immunoglobulin constantdomain may be selected from the group consisting of a human IgM constantdomain, a human IgG4 constant domain, a human IgG1 constant domain, ahuman IgE constant domain, a human IgG2 constant domain, a human IgG3constant domain, and a human IgA constant domain. In another aspect, thebinding protein of the invention further comprises a heavy chainconstant region having an amino acid sequence selected from the groupconsisting of SEQ ID NO:25 and SEQ ID NO:26, additionally a light chainconstant region having an amino acid sequence selected from the groupconsisting of SEQ ID NO:27 and SEQ ID NO:28. In a particular aspect ofthe invention, the binding protein described herein comprises a variableheavy chain comprising an amino acid sequence set forth in SEQ ID NO:6or SEQ ID NO:10; a variable light chain comprising an amino acidsequence set forth in SEQ ID NO:14; a heavy chain constant region havingan amino acid sequence set forth in SEQ ID NO:25; and a light chainconstant region having an amino acid sequence set forth in SEQ ID NO:27.In a further particular aspect of the invention, the binding proteindescribed herein comprises a first amino acid sequence set forth in SEQID NO:46 or SEQ ID NO:47, and a second first amino acid sequence setforth in SEQ ID NO:48.

The binding protein, e.g. the antibody, described herein may furthercomprise a therapeutic agent, an imaging agent, residues capable offacilitating formation of an immunoadhesion molecule and/or anotherfunctional molecule (e.g. another peptide or protein). The imaging agentcan be a radiolabel including but not limited to ³H, ¹⁴C, ³⁵S, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm; an enzyme, a fluorescentlabel, a luminescent label, a bioluminescent label, a magnetic label, orbiotin.

The binding protein of the present invention can be glycosylated.According to one aspect of the invention, the glycosylation pattern is ahuman glycosylation pattern.

In one aspect of the invention, the above-described binding proteinbinds to an Aβ form that comprises the globulomer epitope with which themurine monoclonal antibody m4D10 is reactive (i.e. a targeted Aβ form).In particular the above-described binding proteins bind to amyloid-beta(20-42) globulomer as described herein.

In one aspect of the invention, the binding protein described herein iscapable of modulating a biological function of Aβ(20-42) globulomer. Ina further aspect of the invention, the binding protein described hereinis capable of neutralizing Aβ(20-42) globulomer activity.

The binding protein of the present invention may exist as a crystal. Inone aspect, the crystal is a carrier-free pharmaceutical controlledrelease crystal. In another aspect, the crystallized binding protein hasa greater half life in vivo than its soluble counterpart. In stillanother aspect, the crystallized binding protein retains biologicalactivity after crystallization.

The present invention also provides an isolated nucleic acid encodingany one of the binding proteins disclosed herein. A further embodimentprovides a vector comprising said nucleic acid. Said vector may beselected from the group consisting of pcDNA, pTT (Durocher et al.,Nucleic Acids Research 30(2), 2002), pTT3 (pTT with additional multiplecloning site), pEFBOS (Mizushima and Nagata, Nucleic acids Research18(17), 1990), pBV, pJV, and pBJ.

In another aspect of the invention, a host cell is transformed with thevector disclosed above. According to one embodiment, the host cell is aprokaryotic cell including but not limited to E. coli. In a relatedembodiment, the host cell is a eukaryotic cell selected from the groupcomprising a protist cell, animal cell, plant cell and fungal cell. Theanimal cell may be selected from the group consisting of a mammaliancell, an avian cell and an insect cell. According to one aspect of theinvention, said mammalian cell is selected from the group comprising CHOand COS, said fungal cell is a yeast cell such as Saccharomycescerevisiae, and said insect cell is an insect Sf9 cell.

Further, the invention provides a method of producing a binding proteinas disclosed herein that comprises culturing any one of the host cellsdisclosed herein in a culture medium under conditions and for a timesuitable to produce said binding protein. Another embodiment provides abinding protein of the invention produced according to the methoddisclosed herein. In another embodiment, the invention provides abinding protein produced according to the method disclosed above.

The invention also provides a pharmaceutical composition comprising abinding protein, e.g. an antibody, as disclosed herein and apharmaceutically acceptable carrier.

One embodiment of the invention provides a composition for the releaseof the binding protein described herein wherein the compositioncomprises a formulation which in turn comprises a crystallized bindingprotein, e.g. a crystallized antibody, as disclosed above, and aningredient; and at least one polymeric carrier. In one aspect thepolymeric carrier is a polymer selected from one or more of the groupconsisting of: poly(acrylic acid), poly(cyano-acrylates), poly(aminoacids), poly(anhydrides), poly(depsipeptides), poly(esters), poly(lacticacid), poly(lactic-co-glycolic acid) or PLGA, poly(β-hydroxybutryate),poly(caprolactone), poly(dioxanone); poly(ethylene glycol),poly((hydroxypropyl) methacrylamide), poly((organo)phosphazene),poly(ortho esters), poly(vinyl alcohol), poly(vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof. In another aspect theingredient is selected from the group consisting of: albumin, sucrose,trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin,methoxypolyethylene glycol and polyethylene glycol.

The present invention also relates to a method of inhibiting (i.e.reducing) the activity of Aβ(20-42) globulomer (or any other targeted Aβform) comprising contacting said targeted Aβ form with bindingprotein(s) of the invention such that the activity of said targeted APform is inhibited (i.e. reduced). In a particular embodiment, saidactivity is inhibited in vitro. This method may comprise adding thebinding protein of the invention to a sample, e.g. a sample derived froma subject (e.g., whole blood, cerebrospinal fluid, serum, tissue, etc.)or a cell culture which contains or is suspected to contain a targetedAβ form, in order to inhibit (i.e. reduce) the activity of the Aβ formin the sample. Alternatively, the activity of said targeted Aβ form maybe inhibited (i.e. reduced) in a subject in vivo. Thus, the presentinvention further relates to the binding protein described herein foruse in inhibiting (i.e. reducing) the activity of a targeted Aβ form ina subject comprising contacting said Aβ form with binding protein(s) ofthe invention such that the activity of the Aβ form is inhibited (i.e.reduced).

In a related aspect, the invention provides a method for inhibiting(i.e. reducing) the activity of a targeted Aβ form in a subjectsuffering from a disease or disorder in which the activity of said Aβform is detrimental. In one embodiment, said method comprisesadministering to the subject at least one of the binding proteinsdisclosed herein such that the activity of a targeted AB form in thesubject is inhibited (i.e. reduced). Thus, the invention provides the Aβbinding proteins described herein for use in inhibiting (i.e. reducing)a targeted Aβ form in a subject suffering from a disease or disorder asdescribed herein, wherein at least one of the binding proteins disclosedherein is administered to the subject such that the activity of said Aβform in the subject is inhibited (i.e. reduced).

In a related aspect, the invention provides a method for treating (e.g.,curing, suppressing, ameliorating, delaying or preventing the onset of,or preventing recurrence or relapse of) or preventing a disease ordisorder selected from the group consisting ofAlpha1-antitrypsin-deficiency, C1-inhibitor deficiency angioedema,Antithrombin deficiency thromboembolic disease, Kuru, Creutzfeld-Jacobdisease/scrapie, Bovine spongiform encephalopathy,Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia,Huntington's disease, Spinocerebellar ataxia, Machado-Joseph atrophy,Dentato-rubro-pallidoluysian atrophy, Frontotemporal dementia, Sicklecell anemia, Unstable hemoglobin inclusion-body hemolysis, Drug-inducedinclusion body hemolysis, Parkinson's disease, Systemic AL amyloidosis,Nodular AL amyloidosis, Systemic AA amyloidosis, Prostatic amyloidosis,Hemodialysis amyloidosis, Hereditary (Icelandic) cerebral angiopathy,Huntington's disease, Familial visceral amyloidosis, Familial visceralpolyneuropathy, Familial visceral amyloidosis, Senile systemicamyloidosis, Familial amyloid neurophathy, Familial cardiac amyloidosis,Alzheimer's disease, Down syndrome, Medullary carcinoma thyroid and Type2 diabetes mellitus (T2DM). In a particular embodiment, said disease ordisorder is an amyloidosis such as Alzheimer's disease or Down syndrome.In one embodiment, said method comprising the step of administering anyone of the Aβ binding proteins disclosed herein such that treatment isachieved. In another embodiment, the invention provides a method oftreating a subject suffering from a disease or disorder disclosed hereincomprising the step of administering any one of the Aβ binding proteinsdisclosed herein, concurrent with or after the administration of one ormore additional therapeutic agent(s). Thus, the invention provides theAβ binding proteins disclosed herein for use in treating a subjectsuffering from a disease or disorder disclosed herein comprising thestep of administering any one of the binding proteins disclosed herein,concurrent with or after the administration of one or more additionaltherapeutic agent(s). For instance, the additional therapeutic agent isselected from the group of therapeutic agents listed herein.

The binding proteins disclosed herein and the pharmaceuticalcompositions comprising said binding proteins are administered to asubject by at least one mode selected from parenteral, subcutaneous,intramuscular, intravenous, intraarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

In another embodiment, the present invention provides a method fordetecting a targeted AB form in a sample comprising (i) contacting saidsample with binding protein(s) of the invention and (ii) detectingformation of a complex between said binding protein(s) and elements ofsaid sample, wherein formation or increased formation of the complex inthe sample relative to a control sample indicates the presence of saidAβ form in the sample. The sample may be a biological sample obtainedfrom a subject which is suspected of having a disease or disorder asdisclosed herein (e.g., whole blood, cerebrospinal fluid, serum, tissue,etc.) or a cell culture which contains or is suspected to contain saidAβ form. The control sample does not contain said Aβ form or is obtainedfrom a patient not having a disease as described above. The presence ofa complex between said binding protein(s) and elements of a sampleobtained from a patient suspected of having Alzheimer's diseaseindicates a diagnosis of this disease in said patient.

In an alternative embodiment, the detection of the targeted Aβ form maybe performed in vivo, e.g. by in vivo imaging in a subject. For thispurpose, the binding protein(s) of the invention may be administered toa subject or a control subject under conditions that allow binding ofsaid protein(s) to the targeted Aβ form and detecting formation of acomplex between said binding protein(s) and said Aβ form, whereinformation or increased formation of the complex in the subject relativeto the control subject indicates the presence of said AP form in thesubject. The subject may be a subject which is known or suspected tosuffer from a disorder or disease in which activity of a targeted Aβform is detrimental.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates amino acid sequences (SEQ ID NO:1) of the variablelight chain of humanized 4D10 antibodies comprising Jκ2 and Vκ A17/2-30framework regions. All CDR regions are underlined.

FIG. 2 illustrates amino acid sequences (SEQ ID NO:2) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 (hJH6) andVH3_53 framework regions. All CDR regions are underlined.

FIG. 3 illustrates amino acid sequences (SEQ ID NO:3) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 and VH4_59framework regions. All CDR regions are underlined.

FIG. 4 illustrates the amino acid sequence (SEQ ID NO:4) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 (hJH6) andVH3_53 framework regions. All CDR regions are underlined.

FIG. 5 illustrates the amino acid sequence (SEQ ID NO:5) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 and VH3_53framework regions with VH3 consensus change I12V. All CDR regions areunderlined.

FIG. 6 illustrates the amino acid sequence (SEQ ID NO:6) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 and VH3_53framework regions with VH3 consensus change I12V and frameworkbackmutations A24V, V29L, V48L, S49G, F67L, R71K, N76S and L78V. All CDRregions are underlined.

FIG. 7 illustrates the amino acid sequence (SEQ ID NO:7) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 and VH3_53framework regions with framework backmutations V29L and R71K. All CDRregions are underlined.

FIG. 8 illustrates the amino acid sequence (SEQ ID NO:8) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 and VH4_59framework regions. All CDR regions are underlined.

FIG. 9 illustrates the amino acid sequence (SEQ ID NO:9) of the variableheavy chain of humanized 4D10 antibodies comprising human JH6 and VH4_59framework regions with a Q1E change to prevent N-terminal pyroglutamateformation. All CDR regions are underlined.

FIG. 10 illustrates the amino acid sequence (SEQ ID NO:10) of thevariable heavy chain of humanized 4D10 antibodies comprising human JH6and VH4_59 framework regions with a Q1E change to prevent N-terminalpyroglutamate formation, and framework backmutations G27F, I29L, I37V,I48L, V67L, V71K, N76S and F78V. All CDR regions are underlined.

FIG. 11 illustrates the amino acid sequence (SEQ ID NO:11) of thevariable heavy chain of humanized 4D10 antibodies comprising human JH6and VH4_59 framework regions with a Q1E change to prevent N-terminalpyroglutamate formation, and framework backmutations G27F, I29L andV71K. All CDR regions are underlined.

FIG. 12 illustrate the amino acid sequence (SEQ ID NO:12) of thevariable light chain of humanized 4D10 antibodies comprising Jκ2 and VκA17/2-30 framework regions. All CDR regions are underlined.

FIG. 13 illustrates the amino acid sequence (SEQ ID NO:13) of thevariable light chain of humanized 4D10 antibodies comprising Jκ2 and VκA17/2-30 framework regions with Vκ2 consensus changes S7T, L15P, Q37L,R39K and R45Q. All CDR regions are underlined.

FIG. 14 illustrates the amino acid sequence (SEQ ID NO:14) of thevariable light chain of humanized 4D10 antibodies comprising Jκ2 and VκA17/2-30 framework regions with Vκ2 consensus changes S7T, L15P, Q37L,R39K and R45Q, and framework backmutation F36L. All CDR regions areunderlined.

FIG. 15 illustrates the amino acid sequence (SEQ ID NO:15) of thevariable light chain of humanized 4D10 antibodies comprising Jκ2 and VκA17/2-30 framework regions with Vκ2 consensus changes S7T and Q37L. AllCDR regions are underlined.

FIG. 16 illustrates the amino acid sequence (SEQ ID NO:16) of thevariable light chain of humanized 4D10 antibodies comprising Jκ2 and VκA17/2-30 framework regions with Vκ2 consensus changes S7T, Q37L andR39K. All CDR regions are underlined.

FIG. 17 illustrates an amino acid sequence alignment of the variableheavy chains of murine monoclonal antibody 4D10 (m4D19) and humanized4D10 antibodies (4D10hum) comprising human JH6 (hJH6) and VH353framework regions. All CDR regions are printed in bold letters. X onposition 12 is I or V, X on position 24 is A or V, X on position 29 is Vor L, X on position 48 is V or L, X on position 49 is S or G, X onposition 67 is F or L, X on position 71 is R or K, X on position 76 is Nor S, and X on position 78 is L or V.

FIG. 18 illustrates an amino acid sequence alignment of the variableheavy chains of murine monoclonal antibody 4D10 (m4D19) and humanized4D10 antibodies (4D10hum) comprising human JH6 and VH4_59 frameworkregions. All CDR regions are printed in bold letters. X on position 1 isQ or E, X on position 27 is G or F, X on position 29 is I or L, X onposition 37 is I or V, X on position 48 is I or L, X on position 67 is Vor L, X on position 71 is V or K, X on position 76 is N or S, and X onposition 78 is F or V.

FIG. 19 illustrates an amino acid sequence alignment of the variablelight chains of murine monoclonal antibody 4D10 (m4D19) and humanized4D10 antibodies (4D10hum) comprising Jκ2 and Vκ A17/2-30 frameworkregions. All CDR regions are printed in bold letters. X on position 7 isS or T, X on position 15 is L or P, X on position 41 is F or L, X onposition 42 is Q or L, X on position 44 is R or K, and X on position 50is R or Q.

FIG. 20A and FIG. 20B show platelet factor 4 (PF-4) cross-reaction ofhumanized monoclonal antibodies 4D10hum#1 and 4D10hum#2, human/mousechimeric antibody h1G5 (positive control) and human polyclonal antibodyhIgG1 (negative control) in (A) Cynomolgus monkey plasma and (B) humanplasma, as determined by sandwich-ELISA. Binding of PF-4 to theimmobilized antibodies was detected.

FIG. 21A and FIG. 21B show platelet factor 4 (PF-4) cross-reaction ofhumanized monoclonal antibodies 4D10hum#1 and 4D10hum#2, human/mousechimeric antibody h1G5 (positive control) and human polyclonal antibodyhIgG1 (negative control) in (A) Cynomolgus monkey plasma and (B) humanplasma, as determined by aligned sandwich-ELISA. The antibodies werecaptured on the plate by immobilized anti-mouse IgG. Binding of PF-4 tothe captured antibodies was detected.

FIG. 22A and FIG. 22B show platelet factor 4 (PF-4) cross-reaction ofmurine monoclonal antibodies m4D10 and m1G5, anti human PF-4 antibody(positive control) and IgG2a (negative control) in (A) Cynomolgus monkeyplasma and (B) human plasma, as determined by sandwich-ELISA. Binding ofPF-4 to the immobilized antibodies was detected.

FIG. 23A and FIG. 23B show platelet factor 4 (PF-4) cross-reaction ofmurine monoclonal antibodies m4D10 and m1G5, anti human PF-4 antibody(positive control) and IgG2a (negative control) in (A) Cynomolgus monkeyplasma and (B) human plasma, as determined by aligned sandwich-ELISA.The antibodies were captured on the plate by immobilized anti-mouse IgG.Binding of PF-4 to the captured antibodies was detected.

FIG. 24 illustrates the amino acid sequence (SEQ ID NO:46) of the heavychain of a humanized 4D10 antibody comprising human JH6 and VH3_53framework regions with VH3 consensus change I12V and frameworkbackmutations A24V, V29L, V48L, S49G, F67L, R71K, N76S and L78V; and anIg gamma-1 constant region. All CDR regions are underlined.

FIG. 25 illustrates the amino acid sequence (SEQ ID NO:47) of the heavychain of a humanized 4D10 antibody comprising human JH6 and VH4_59framework regions with a Q1E change to prevent N-terminal pyroglutamateformation, and framework backmutations G27F, I29L, I37V, I48L, V67L,V71K, N76S and F78V; and an Ig gamma-1 constant region. All CDR regionsare underlined.

FIG. 26 illustrates the amino acid sequence (SEQ ID NO:48) of the lightchain of a humanized 4D10 antibody comprising Jκ2 and Vκ A17/2-30framework regions with Via consensus changes S7T, L15P, Q37L, R39K andR45Q, and framework backmutation F36L; and an Ig kappa constant region.All CDR regions are underlined.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics, protein and nucleic acid chemistry, and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

The present invention pertains to Aβ binding proteins, particularlyanti-Aβ antibodies or an Aβ binding portion thereof, particularly thosebinding to Aβ(20-42) globulomer. These AP binding proteins are capableof discriminating not only other forms of Aβ peptides, particularlymonomers and fibrils, but also untruncated forms of Aβ globulomers.Thus, the present invention relates to an Aβ binding protein having abinding affinity to an Aβ(20-42) globulomer that is greater than thebinding affinity of this Aβ binding protein to an Aβ(1-42) globulomer.

The term “Aβ(X-Y)” as used herein refers to the amino acid sequence fromamino acid position X to amino acid position Y of the human amyloid beta(Aβ) protein including both X and Y, in particular to the amino acidsequence from amino acid position X to amino acid position Y of theamino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT (SEQID NO:29) (corresponding to amino acid positions 1 to 43) or any of itsnaturally occurring variants, in particular those with at least onemutation selected from the group consisting of A2T, H6R, D7N, A21G(“Flemish”), E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N(“Iowa”), A42T and A42V wherein the numbers are relative to the start ofthe Aβ peptide, including both position X and position Y or a sequencewith up to three additional amino acid substitutions none of which mayprevent globulomer formation. According to one aspect, there are noadditional amino acid substitutions in the portion from amino acid 12 orX, whichever number is higher, to amino acid 42 or Y, whichever numberis lower. According to another aspect, there are no additional aminoacid substitutions in the portion from amino acid 20 or X, whichevernumber is higher, to amino acid 42 or Y, whichever number is lower.According to another aspect, there are no additional amino acidsubstitutions in the portion from amino acid 20 or X, whichever numberis higher, to amino acid 40 or Y, whichever number is lower. An“additional” amino acid substitution herein is any deviation from thecanonical sequence that is not found in nature.

More specifically, the term “Aβ(1-42)” as used herein refers to theamino acid sequence from amino acid position 1 to amino acid position 42of the human Aβ protein including both 1 and 42, in particular to theamino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQID NO:30) or any of its naturally occurring variants, in particularthose with at least one mutation selected from the group consisting ofA2T, H6R, D7N, A21G (“Flemish”), E22G (“Arctic”), E22Q (“Dutch”), E22K(“Italian”), D23N (“Iowa”), A42T and A42V wherein the numbers arerelative to the start of the AP peptide, including both 1 and 42 or asequence with up to three additional amino acid substitutions none ofwhich may prevent globulomer formation. According to one aspect, thereare no additional amino acid substitutions in the portion from aminoacid 20 to amino acid 42. Likewise, the term “Aβ(1-40)” as used hereinrefers to the amino acid sequence from amino acid position 1 to aminoacid position 40 of the human Aβ protein including both 1 and 40, inparticular to the amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGAIIGLMVGGVV (SEQ ID NO:31) or any of its naturally occurring variants, inparticular those with at least one mutation selected from the groupconsisting of A2T, H6R, D7N, A21G (“Flemish”), E22G (“Arctic”), E22Q(“Dutch”), E22K (“Italian”), and D23N (“Iowa”) wherein the numbers arerelative to the start of the Aβ peptide, including both 1 and 40 or asequence with up to three additional amino acid substitutions none ofwhich may prevent globulomer formation. According to one aspect, thereare no additional amino acid substitutions in the portion from aminoacid 20 to amino acid 40.

More specifically, the term “Aβ(12-42)” as used herein refers to theamino acid sequence from amino acid position 12 to amino acid position42 of the human Aβ protein including both 12 and 42, in particular tothe amino acid sequence VHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ IDNO:32) or any of its naturally occurring variants, in particular thosewith at least one mutation selected from the group consisting of A21G(“Flemish”), E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N(“Iowa”), A42T and A42V wherein the numbers are relative to the start ofthe Aβ peptide, including both 12 and 42 or a sequence with up to threeadditional amino acid substitutions none of which may prevent globulomerformation. According to one aspect, there are no additional amino acidsubstitutions in the portion from amino acid 20 to amino acid 42.Likewise, the term “Aβ(20-42)” as used herein refers to the amino acidsequence from amino acid position 20 to amino acid position 42 of thehuman amyloid β protein including both 20 and 42, in particular to theamino acid sequence F AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO:33) or any ofits naturally occurring variants, in particular those with at least onemutation selected from the group consisting of A21G (“Flemish”), E22G(“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T andA42V wherein the numbers are relative to the start of the AP peptide,including both 20 and 42 or a sequence with up to three additional aminoacid substitutions none of which may prevent globulomer formation.According to one aspect, there are any additional amino acidsubstitutions.

The term “Aβ(X-Y) globulomer” (Aβ(X-Y) globular oligomer) as used hereinrefers to a soluble, globular, non-covalent association of Aβ(X-Y)peptides as defined above, possessing homogeneity and distinct physicalcharacteristics. According to one aspect, Aβ(X-Y) globulomers arestable, non-fibrillar, oligomeric assemblies of Aβ(X-Y) peptides whichare obtainable by incubation with anionic detergents. In contrast tomonomer and fibrils, these globulomers are characterized by definedassembly numbers of subunits (e.g. early assembly forms with 4-6subunits, “oligomers A”; and late assembly forms with 12-14 subunits,“oligomers B”; as described in WO2004/067561). The globulomers have a3-dimensional globular type structure (“molten globule”, see Barghorn etal., J Neurochem 95: 834-847, 2005). They may be further characterizedby one or more of the following features:

-   -   cleavability of N-terminal amino acids X-23 with promiscuous        proteases (such as thermolysin or endoproteinase GluC) yielding        truncated forms of globulomers;    -   non-accessibility of C-terminal amino acids 24-Y with        promiscuous proteases and antibodies;    -   truncated forms of these globulomers maintain the 3-dimensional        core structure of said globulomers with a better accessibility        of the core epitope Aβ(20-Y) in its globulomer conformation.

According to the invention and in particular for the purpose ofassessing the binding affinities of the Aβ binding proteins of thepresent invention, the term “Aβ(X-Y) globulomer” here refers inparticular to a product which is obtainable by a process as described inWO2004/067561, which is incorporated herein by reference. Said processcomprises unfolding a natural, recombinant or synthetic Aβ(X-Y) peptideor a derivative thereof; exposing the at least partially unfoldedAβ(X-Y) peptide or derivative thereof to a detergent, reducing thedetergent action and continuing incubation.

For the purpose of unfolding the peptide, hydrogen bond-breaking agentssuch as, for example, hexafluoroisopropanol (HFIP) may be allowed to acton the protein. Times of action of a few minutes, for example about 10to 60 minutes, are sufficient when the temperature of action is fromabout 20 to 50° C. and in particular about 35 to 40° C. Subsequentdissolution of the residue evaporated to dryness, e.g. in concentratedform, in suitable organic solvents miscible with aqueous buffers, suchas, for example, dimethyl sulfoxide (DMSO), results in a suspension ofthe at least partially unfolded peptide or derivative thereof, which canbe used subsequently. If required, the stock suspension may be stored atlow temperature, for example at about 20° C., for an interim period.Alternatively, the peptide or the derivative thereof may be taken up inslightly acidic, e.g. aqueous, solution, for example, an about 10 mMaqueous HCl solution. After an incubation time of usually a few minutes,insoluble components are removed by centrifugation. A few minutes at10,000 g is expedient. These method steps can be carried out at roomtemperature, i.e. a temperature in the range from 20 to 30° C. Thesupernatant obtained after centrifugation contains the Aβ(X-Y) peptideor the derivative thereof and may be stored at low temperature, forexample at about −20° C., for an interim period. The following exposureto a detergent relates to the oligomerization of the peptide or thederivative thereof to give an intermediate type of oligomers (in WO2004/067561 referred to as oligomers A). For this purpose, a detergentis allowed to act on the at least partially unfolded peptide orderivative thereof until sufficient intermediate oligomer has beenproduced. Preference is given to using ionic detergents, in particularanionic detergents.

According to a particular embodiment, a detergent of the formula (I):R—X,is used, in which the radical R is unbranched or branched alkyl havingfrom 6 to 20, e.g. 10 to 14, carbon atoms or unbranched or branchedalkenyl having from 6 to 20, e.g. 10 to 14, carbon atoms, the radical Xis an acidic group or salt thereof, with X being selected, e.g., fromamong —COO-M⁺, —SO₃-M⁺, and especially —OSO₃-M⁺ and M⁺ is a hydrogencation or an inorganic or organic cation selected from, e.g., alkalimetal and alkaline earth metal cations and ammonium cations.Advantageous are detergents of the formula (I), in which R is unbranchedalkyl of which alk-1-yl radicals must be mentioned in particular. Forexample, sodium dodecyl sulfate (SDS), lauric acid, the sodium salt ofthe detergent lauroylsarcosin (also known as sarkosyl NL-30 or Gardol®)and oleic acid can be used advantageously. The time of detergent actionin particular depends on whether (and if yes, to what extent) thepeptide or the derivative thereof subjected to oligomerization hasunfolded. If, according to the unfolding step, the peptide or derivativethereof has been treated beforehand with a hydrogen bond-breaking agent,i.e. in particular with hexafluoroisopropanol, times of action in therange of a few hours, advantageously from about 1 to 20 and inparticular from about 2 to 10 hours, are sufficient when the temperatureof action is about 20 to 50° C. and in particular about 35 to 40° C. Ifa less unfolded or an essentially not unfolded peptide or derivativethereof is the starting point, correspondingly longer times of actionare expedient. If the peptide or the derivative thereof has beenpretreated, for example, according to the procedure indicated above asan alternative to the HFIP treatment or said peptide or derivativethereof is directly subjected to oligomerization, times of action in therange from about 5 to 30 hours and in particular from about 10 to 20hours are sufficient when the temperature of action is about 20 to 50°C. and in particular about 35 to 40° C. After incubation, insolublecomponents are advantageously removed by centrifugation. A few minutesat 10,000 g is expedient. The detergent concentration to be chosendepends on the detergent used. If SDS is used, a concentration in therange from 0.01 to 1% by weight, e.g. from 0.05 to 0.5% by weight, forexample of about 0.2% by weight, proves expedient. If lauric acid oroleic acid are used, somewhat higher concentrations are expedient, forexample in a range from 0.05 to 2% by weight, e.g. from 0.1 to 0.5% byweight, for example of about 0.5% by weight. The detergent action shouldtake place at a salt concentration approximately in the physiologicalrange. Thus, in particular NaCl concentrations in the range from 50 to500 mM, e.g. from 100 to 200 mM or at about 140 mM are expedient. Thesubsequent reduction of the detergent action and continuation ofincubation relates to a further oligomerization to give the Aβ(X-Y)globulomer of the invention (in WO2004/067561 referred to as oligomersB). Since the composition obtained from the preceding step regularlycontains detergent and a salt concentration in the physiological rangeit is then expedient to reduce detergent action and also the saltconcentration. This may be carried out by reducing the concentration ofdetergent and salt, for example, by diluting, expediently with water ora buffer of lower salt concentration, for example Tris-HCl, pH 7.3.Dilution factors in the range from about 2 to 10, advantageously in therange from about 3 to 8 and in particular of about 4, have provedsuitable. The reduction in detergent action may also be achieved byadding substances which can neutralize said detergent action. Examplesof these include substances capable of complexing the detergents, likesubstances capable of stabilizing cells in the course of purificationand extraction measures, for example particular EO/PO block copolymers,in particular the block copolymer under the trade name Pluronic® F 68.Alkoxylated and, in particular, ethoxylated alkyl phenols such as theethoxylated t-octylphenols of the Triton® X series, in particularTriton® X100, 3-(3-cholamidopropyldimethylammonio)-1-propanesulfonate(CHAPS®) or alkoxylated and, in particular, ethoxylated sorbitan fattyesters such as those of the Tween® series, in particular Tween® 20, inconcentration ranges around or above the particular critical micelleconcentration, may be equally used. Subsequently, the solution isincubated until sufficient Aβ(X-Y) globulomer of the invention has beenproduced. Times of action in the range of several hours, e.g. in therange from about 10 to 30 hours or in the range from about 15 to 25hours, are sufficient when the temperature of action is about 20 to 50°C. and in particular about 35 to 40° C. The solution may then beconcentrated and possible residues may be removed by centrifugation.Here too, a few minutes at 10,000 g proves expedient. The supernatantobtained after centrifugation contains an Aβ(X-Y) globulomer of theinvention. An Aβ(X-Y) globulomer of the invention can be finallyrecovered in a manner known per se, e.g. by ultrafiltration, dialysis,precipitation or centrifugation. For example, electrophoretic separationof the Aβ(X-Y) globulomers under denaturing conditions, e.g. bySDS-PAGE, may produce a double band (e.g. with an apparent molecularweight of 38/48 kDa for Aβ(1-42)), and upon glutardialdehyde treatmentof the globulomers before separation these two bands can merge into one.Size exclusion chromatography of the globulomers may result in a singlepeak (e.g. corresponding to a molecular weight of approximately 100 kDafor Aβ(1-42) globulomer or of approximately 60 kDa for glutardialdehydecross-linked Aβ(1-42) globulomer), respectively. Starting out fromAβ(1-42) peptide, Aβ(12-42) peptide, and Aβ(20-42) peptide saidprocesses are in particular suitable for obtaining Aβ(1-42) globulomers,Aβ(12-42) globulomers, and Aβ(20-42) globulomers.

In a particular embodiment of the invention, Aβ(X-Y) globulomers whereinX is selected from the group consisting of the numbers 2 . . . 24 and Yis as defined above, are those which are obtainable by truncatingAβ(1-Y) globulomers into shorter forms wherein X is selected from thegroup consisting of the numbers 2 . . . 24, for example with X being 20or 12, and Y is as defined above, which can be achieved by treatmentwith appropriate proteases. For instance, an Aβ(20-42) globulomer can beobtained by subjecting an Aβ(1-42) globulomer to thermolysinproteolysis, and an Aβ(12-42) globulomer can be obtained by subjectingan Aβ(1-42) globulomer to endoproteinase GluC proteolysis. When thedesired degree of proteolysis is reached, the protease is inactivated ina generally known manner. The resulting globulomers may then be isolatedfollowing the procedures already described herein and, if required,processed further by further work-up and purification steps. A detaileddescription of said processes is disclosed in WO2004/067561, which isincorporated herein by reference.

For the purposes of the present invention, an Aβ(1-42) globulomer is, inparticular, the Aβ(1-42) globulomer as described in Example 1a below; anAβ(20-42) globulomer is in particular the Aβ(20-42) globulomer asdescribed in Example 1b below, and an Aβ(12-42) globulomer is inparticular the Aβ(12-42) globulomer as described in Example 1c below.According to one aspect of the invention, the globulomer shows affinityto neuronal cells and/or exhibits neuromodulating effects.

According to another aspect of the invention, the globulomer consists of11 to 16, e.g. of 12 to 14 Aβ(X-Y) peptides. According to another aspectof the invention, the term “Aβ(X-Y) globulomer” herein refers to aglobulomer consisting essentially of Aβ(X-Y) subunits, where for exampleon average at least 11 of 12 subunits are of the Aβ(X-Y) type, or lessthan 10% of the globulomers comprise any non-Aβ(X-Y) peptides, or thecontent of non-Aβ(X-Y) peptides is below the detection threshold. Morespecifically, the term “Aβ(1-42) globulomer” herein refers to aglobulomer consisting essentially of Aβ(1-42) units as defined above;the term “Aβ(12-42) globulomer” herein refers to a globulomer consistingessentially of Aβ(12-42) units as defined above; and the term “Aβ(20-42)globulomer” herein refers to a globulomer consisting essentially ofAβ(20-42) units as defined above.

The term “cross-linked Aβ(X-Y) globulomer” herein refers to a moleculeobtainable from an Aβ(X-Y) globulomer as described above bycross-linking, e.g. by chemically cross-linking, aldehyde cross-linking,glutardialdehyde cross-linking, of the constituent units of theglobulomer. In another aspect of the invention, a cross-linkedglobulomer is essentially a globulomer in which the units are at leastpartially joined by covalent bonds, rather than being held together bynon-covalent interactions only. For the purposes of the presentinvention, a cross-linked Aβ(1-42) globulomer is in particular thecross-linked Aβ(1-42) oligomer as described in Example 1d below.

The term “Aβ(X-Y) globulomer derivative” herein refers in particular toa globulomer that is labelled by being covalently linked to a group thatfacilitates detection, for example a fluorophore, e.g. fluoresceinisothiocyanate, phycoerythrin, Aequorea victoria fluorescent protein,Dictyosoma fluorescent protein or any combination or fluorescence-activederivative thereof; a chromophore; a chemoluminophore, e.g. luciferase,in particular Photinus pyralis luciferase, Vibrio fischeri luciferase,or any combination or chemoluminescence-active derivative thereof; anenzymatically active group, e.g. peroxidase, e.g. horseradishperoxidase, or any enzymatically active derivative thereof; anelectron-dense group, e.g. a heavy metal containing group, e.g. a goldcontaining group; a hapten, e.g. a phenol derived hapten; a stronglyantigenic structure, e.g. peptide sequence predicted to be antigenic,e.g. predicted to be antigenic by the algorithm of Kolaskar andTongaonkar; an aptamer for another molecule; a chelating group, e.g.hexahistidinyl; a natural or nature-derived protein structure mediatingfurther specific protein-protein interactions, e.g. a member of thefos/jun pair; a magnetic group, e.g. a ferromagnetic group; or aradioactive group, e.g. a group comprising 1H, 14C, 32P, 35S or 125I orany combination thereof; or to a globulomer flagged by being covalentlyor by non-covalent high-affinity interaction linked to a group thatfacilitates inactivation, sequestration, degradation and/orprecipitation, for example flagged with a group that promotes in vivodegradation such as ubiquitin, this flagged oligomer being, e.g.,assembled in vivo; or to a globulomer modified by any combination of theabove. Such labelling and flagging groups and methods for attaching themto proteins are known in the art. Labelling and/or flagging may beperformed before, during or after globulomerisation. In another aspectof the invention, a globulomer derivative is a molecule obtainable froma globulomer by a labelling and/or flagging reaction. Correspondingly,term “Aβ(X-Y) monomer derivative” here refers in particular to an Aβmonomer that is labelled or flagged as described for the globulomer.

In a further aspect of the invention, the binding proteins describedherein bind to the Aβ(20-42) globulomer with a high affinity, forinstance with a dissociation constant (K_(D)) of at most about 10⁻⁶M; atmost about 10⁻⁷ M; at most about 10⁻⁸ M; at most about 10⁻⁹ M; at mostabout 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about 10⁻¹² M; and at most10⁻¹³ M. In one aspect the on-rate constant (k_(on)) of the bindingprotein described herein to Aβ(20-42) globulomer is selected from thegroup consisting of: at least about 10² M⁻¹ s⁻¹; at least about 10³ M⁻¹s⁻¹; at least about 10⁴ M⁻¹ s⁻¹; at least about 10⁵ M⁻¹ s⁻¹; and atleast about 10⁶ M^(−l) s⁻¹; as measured by surface plasmon resonance. Inanother aspect, the binding proteins have an off-rate constant (k_(off))to Aβ(20-42) globulomer selected from the group consisting of: at mostabout 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at most about 10⁻⁵ s⁻¹; and atmost about 10⁻⁶ S⁻¹, as measured by surface plasmon resonance. In aparticular aspect of the invention, the binding proteins describedherein bind to the Aβ(20-42) globulomer with a dissociation constantfrom 1×10⁻⁹ to 1×10⁻¹⁰ M. In a further particular aspect of theinvention, the on-rate constant (k_(on)) of the binding proteindescribed herein to Aβ(20-42) globulomer is from 1×10⁵ to 1×10⁶ M⁻¹ s⁻¹.In a further particular aspect of the invention, the binding proteinsdescribed herein have an off-rate constant (k_(off)) to Aβ(20-42)globulomer from 8×10⁻⁵ to 8×10⁶M⁻¹ s⁻¹.

In another aspect of the invention, the binding affinity of the bindingproteins described herein to Aβ(20-42) globulomer is greater than to anAβ(1-42) globulomer.

The term “greater affinity” herein refers to a degree of interactionwhere the equilibrium between unbound Aβ binding protein and unbound Aβglobulomer on the one hand and AP binding protein-globulomer complex onthe other is further in favour of the Aβ binding protein-globulomercomplex. Likewise, the term “smaller affinity” here refers to a degreeof interaction where the equilibrium between unbound Aβ binding proteinand unbound AP globulomer on the one hand and Aβ bindingprotein-globulomer complex on the other is further in favour of theunbound Aβ binding protein and unbound Aβ globulomer. The term “greateraffinity” is synonymous with the term “higher affinity” and term“smaller affinity” is synonymous with the term “lower affinity”.

In a related aspect of the invention, the binding affinity of thebinding proteins described herein to Aβ(20-42) globulomer is at least 2times (e.g., at least 3 or at least 5 times), at least 10 times (e.g.,at least 20 times, at least 30 times or at least 50 times), at least 100times (e.g., at least 200 times, at least 300 times or at least 500times), and at least 1,000 times (e.g., at least 2,000 times, at least3,000 times or at least 5000 times), at least 10,000 times (e.g., atleast 20,000 times, at least 30,000 times or at least 50,000 times), orat least 100,000 times greater than the binding affinity of the bindingprotein to the Aβ(1-42) globulomer.

In still a further aspect of the invention, the binding proteinsdescribed herein bind to the Aβ(12-42) globulomer with a relatively highaffinity, for instance with a dissociation constant (K_(D)) of at mostabout 10⁻⁶ M; at most about 10⁻⁷ M; at most about 10⁻⁸ M; at most about10⁻⁹ M; at most about 10⁻¹⁰ M; at most about 10⁻¹¹ M; at most about10⁻¹² M; and at most 10⁻¹³ M. In one aspect the on-rate constant(k_(on)) of the binding protein described herein to Aβ(12-42) globulomeris selected from the group consisting of: at least about 10² M⁻¹ s⁻¹; atleast about 10³ M⁻¹ s⁻¹; at least about 10⁴ M⁻¹ s⁻¹; at least about 10⁵M⁻¹ s⁻¹; and at least about 10⁶ M⁻¹ s⁻¹; as measured by surface plasmonresonance. In another aspect, the binding proteins have an off-rateconstant (k_(off)) to Aβ(12-42) globulomer selected from the groupconsisting of: at most about 10⁻³ s⁻¹; at most about 10⁻⁴ s⁻¹; at mostabout 10⁻⁵ S⁻¹; and at most about 10⁻⁶ S⁻¹, as measured by surfaceplasmon resonance.

In a related aspect of the invention, the binding affinity of thebinding proteins described herein to Aβ(20-42) globulomer is about 1.1to 3 times greater than the binding affinity of the binding proteins toAβ(12-42) globulomer.

According to one aspect, the Aβ binding proteins of the presentinvention bind to at least one Aβ globulomer, as defined above, and havea comparatively smaller affinity for at least one non-globulomer form ofAP. Aβ binding proteins of the present invention with a comparativelysmaller affinity for at least one non-globulomer form of Aβ than for atleast one Aβ globulomer include Aβ binding protein with a bindingaffinity to the Aβ(20-42) globulomer that is greater than to an Aβ(1-42)monomer. According to an alternative or additional aspect of theinvention, the binding affinity of the Aβ binding protein to theAβ(20-42) globulomer is greater than to an Aβ(1-40) monomer. Inparticular, the affinity of the Aβ binding proteins to the Aβ(20-42)globulomer is greater than its affinity to both the Aβ(1-40) and theAβ(1-42) monomer.

The term “Aβ(X-Y) monomer” as used herein refers to the isolated form ofthe Aβ(X-Y) peptide, in particular to a form of the Aβ(X-Y) peptidewhich is not engaged in essentially non-covalent interactions with otherAβ peptides. Practically, the Aβ(X-Y) monomer is usually provided in theform of an aqueous solution. In a particular embodiment of theinvention, the aqueous monomer solution contains 0.05% to 0.2%, e.g.about 0.1% NH₄OH. In another particular embodiment of the invention, theaqueous monomer solution contains 0.05% to 0.2%, e.g. about 0.1% NaOH.When used (for instance for determining the binding affinities of the Aβbinding proteins of the present invention), it may be expedient todilute said solution in an appropriate manner. Further, it is usuallyexpedient to use said solution within 2 hours, in particular within 1hour, and especially within 30 minutes after its preparation.

More specifically, the term “Aβ(1-40) monomer” here refers to anAβ(1-40) monomer preparation as described herein, and the term “Aβ(1-42)monomer” here refers to an Aβ(1-42) preparation as described herein.

Expediently, the Aβ binding proteins of the present invention bind toone or both monomers with low affinity, for example with a K_(D) of1×10⁻⁸ M or smaller affinity, e.g. with a K_(D) of 3×10⁻⁸ M or smalleraffinity, with a K_(D) of 1×10⁻⁷ M or smaller affinity, e.g. with aK_(D) of 3×10⁻⁷ M or smaller affinity, or with a K_(D) of 1×10⁻⁶ M orsmaller affinity, e.g. with a K_(D) of 3×10⁻⁵ M or smaller affinity, orwith a K_(D) of 1×10⁻⁵ M or smaller affinity.

According to one aspect of the invention, the binding affinity of the Aβbinding proteins of the present invention to the Aβ(20-42) globulomer isat least 2 times, e.g. at least 3 times or at least 5 times, at least 10times, e.g. at least 20 times, at least 30 times or at least 50 times,at least 100 times, e.g. at least 200 times, at least 300 times or atleast 500 times, at least 1,000 times, e.g. at least 2,000 times, atleast 3,000 times or at least 5,000 times, at least 10,000 times, e.g.at least 20,000 times, at least 30,000 or at least 50,000 times, or atleast 100,000 times greater than the binding affinity of the Aβ bindingproteins to one or both monomers.

Aβ binding proteins of the present invention having a comparativelysmaller affinity for at least one non-globulomer form of Aβ than for atleast one Aβ globulomer further include AP binding proteins having abinding affinity to the Aβ(20-42) globulomer that is greater than toAβ(1-42) fibrils. According to an alternative or additional aspect ofthe invention, the binding affinity of the Aβ binding proteins to theAβ(20-42) globulomer is greater than to Aβ(1-40) fibrils. According toone particular embodiment, the invention relates to Aβ binding proteinshaving a binding affinity to the Aβ(20-42) globulomer which is greaterthan their binding affinity to both Aβ(1-40) and Aβ(1-42) fibrils.

The term “fibril” herein refers to a molecular structure that comprisesassemblies of non-covalently associated, individual Aβ(X-Y) peptides,which show fibrillary structure in the electron microscope, which bindCongo red and then exhibit birefringence under polarized light and whoseX-ray diffraction pattern is a cross-β structure. In another aspect ofthe invention, a fibril is a molecular structure obtainable by a processthat comprises the self-induced polymeric aggregation of a suitable Aβpeptide in the absence of detergents, e.g. in 0.1 M HCl, leading to theformation of aggregates of more than 24 or more than 100 units. Thisprocess is well known in the art. Expediently, Aβ(X-Y) fibrils are usedin the form of an aqueous solution. In a particular embodiment of theinvention, the aqueous fibril solution is made by dissolving the Aβpeptide in 0.1% NH₄OH, diluting it 1:4 with 20 mM NaH₂PO₄, 140 mM NaCl,pH 7.4, followed by readjusting the pH to 7.4, incubating the solutionat 37° C. for 20 h, followed by centrifugation at 10,000 g for 10 minand resuspension in 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4. The term“Aβ(X-Y) fibril” herein also refers to a fibril comprising Aβ(X-Y)subunits where, e.g., on average, at least 90% of the subunits are ofthe Aβ(X-Y) type, at least 98% of the subunits are of the Aβ(X-Y) typeor the content of non-Aβ(X-Y) peptides is below the detection threshold.More specifically, the term “Aβ(1-42) fibril” herein refers to aAβ(1-42) fibril preparation as described in Example 3.

Expediently, the Aβ binding proteins of the present invention bind toone or both fibrils with low affinity, for example with a K_(D) of1×10⁻⁸ M or smaller affinity, e.g. with a K_(D) of 3×10⁻⁸ M or smalleraffinity, with a K_(D) of 1×10⁻⁷ M or smaller affinity, e.g. with aK_(D) of 3×10⁻⁷ M or smaller affinity, or with a K_(D) of 1×10⁻⁶ M orsmaller affinity, e.g. with a K_(D) of 3×10⁻⁵ M or smaller affinity, orwith a K_(D) of 1×10⁻⁵ M or smaller affinity.

According to one aspect of the invention, the binding affinity of the Aβbinding proteins of the present invention to the Aβ(20-42) globulomer isat least 2 times, e.g. at least 3 times or at least 5 times, at least 10times, e.g. at least 20 times, at least 30 times or at least 50 times,at least 100 times, e.g. at least 200 times, at least 300 times or atleast 500 times, at least 1,000 times, e.g. at least 2,000 times, atleast 3,000 times or at least 5,000 times, at least 10,000 times, e.g.at least 20,000 times, at least 30,000 or at least 50,000 times, or atleast 100,000 times greater than the binding affinity of the Aβ bindingproteins to one or both fibrils.

According to a particular embodiment, the present invention relates toAβ binding proteins having a comparatively smaller affinity for both themonomeric and fibrillary forms of AP than for at least one Aβglobulomer, in particular Aβ(20-42) globulomer. These Aβ bindingproteins sometimes are referred to as globulomer-specific Aβ bindingproteins.

The binding proteins of the present invention, e.g. humanized antibody4D10 (4D10hum), include globulomer-specific binding proteins recognizingpredominantly Aβ(20-42) globulomer forms and not standard preparationsof Aβ(1-40) monomers, Aβ(1-42) monomers, Aβ-fibrils or sAPP (i.e.soluble Aβ precursor) in contrast to, for example, competitor antibodiessuch as m266 and 3D6. Such specificity for globulomers is importantbecause specifically targeting the globulomer form of Aβ with humanized4D10 will: 1) avoid targeting insoluble amyloid deposits, binding towhich may account for inflammatory side effects observed duringimmunizations with insoluble AB; 2) spare Aβ monomer and APP that arereported to have precognitive physiological functions (Plan et al., JNeurosci 23: 5531-5535, 2003; and 3) increase the bioavailability of theantibody, as it would not be shaded or inaccessible through extensivebinding to insoluble deposits.

PF-4 is a small, 70-amino acid cytokine that belongs to the CXCchemokine family and is also known as chemokine (C—X—C motif) ligand 4(CXCL4). PF-4 is released from alpha-granules of activated plateletsduring platelet aggregation, and promotes blood coagulation bymoderating the effects of heparin-like molecules. Due to thesefunctions, it is predicted to be involved in wound repair andinflammation (Eismann et al., Blood 76(2): 336-44, 1990). PF-4 isusually found in a complex with proteoglycan and can form complexes withthe anticoagulant heparin which is in use as pharmacological treatmentof thrombosis. It has a well described pathological function inheparin-induced thrombocytopenia (HIT), an idiosyncratic autoimmunereaction to the administration of the anticoagulant heparin (Warkentin,N. Engl. J. Med. 356(9): 891-3, 2007), wherein the heparin:PF4 complexis the antigen. PF4 autoantibodies have also been found in patients withthrombosis and features resembling HIT but no prior administration ofheparin (Warkentin et al., Am. J. Med. 121(7): 632-6, 2008).Heparin-induced thrombocytopenia is characterized by the development ofthrombocytopenia (a low platelet count), and in addition HIT predisposesto thrombosis. In view of these functions and involvement of PF-4 inpathological processes it can be concluded that the administration ofbinding proteins (e.g. antibodies) showing binding (e.g.cross-reactivity) to the PF-4 present in a subject may affect said PF-4functions and thus result in adverse (side) effects. The degree andnature of such adverse effects may vary depending on parameters such aslocation and size of the epitope on PF-4, binding strength and nature ofthe respective binding protein.

According to one aspect of the invention, the binding proteins of thepresent invention do show no or low binding to platelet factor 4 (PF-4).Said cross-reaction to PF-4 may be evaluated by using standardized invitro immunoassays such as ELISA, dot blot or BIAcore analyses.

According to a particular embodiment, the cross-reaction to PF-4 of abinding protein defined herein refers to ratio of values for saidbinding protein and a reference anti-PF-4 antibody obtained by (i)performing a sandwich-ELISA with a ˜1:3 dilution series of human orcynomolgus plasma from about 1:3.16 to about 1:3160 (final plasmadilution) (e.g. as described in examples 3.1 and 3.2), (ii) plottingdetected signal (y-axis) against log-transformed plasma dilutions(x-axis), and (iii) determining the area under the curve (AUC, or totalpeak area) from these non-curve fitted data in the measured range (finalplasma dilutions from about 1:3.16 to about 1:3160). According to aparticular embodiment of the invention, determining the cross-reactionto PF-4 by sandwich-ELISA comprises the following: a certain amount ofthe binding protein under investigation or the reference anti-PF-4antibody or, expediently, an appropriate dilution thereof, for instance100 μl of a 10 μg/ml binding protein or antibody solution in 100 mMsodium hydrogen carbonate, pH 9.6, is used for coating wells of aprotein adsorbing microtiter plate; the plate is then washed, blocked,and washed again; then contacted with a ˜1:3 dilution series ofcynomolgus or human plasma, e.g. human plasma spiked with human PF-4,from about 1:3.16 to about 1:3160 (final plasma dilution) followed bydetection of the PF-4 bound to each well, e.g. by means of a primaryPF-4 specific antibody, an enzyme-conjugated secondary antibody and acolorimetric reaction.

A “reference anti-PF-4 antibody”, as used herein, is an antibody, inparticular a monoclonal antibody, that is specifically reactive withPF-4, in particular human (HPF4). Such an antibody is obtainable byproviding an antigen comprising human PF-4, for instance human PF-4having amino acid sequenceEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAP LYKKIIKKLLES(SEQ ID NO:70), exposing an antibody repertoire to said antigen andselecting from said antigen repertoire an antibody which bindsspecifically to human PF-4. The antibody may optionally be affinitypurified using the immunogen (human PF-4). Such reference anti-PF4antibodies are commercially available, for example, monoclonal anti-HPF4antibody, Abcam cat. no.: ab49735.

According to another particular embodiment, the cross-reaction to PF-4of a binding protein defined herein refers to ratio of AUC values forsaid binding protein and a reference anti-PF-4 antibody obtained by (i)performing an aligned sandwich-ELISA with human or cynomolgus plasma and˜1:3 dilution series of binding protein and reference anti-PF-4 antibodyfrom about 10 ng/ml to about 10000 ng/ml (final concentration) (e.g. asdescribed in examples 3.3 and 3.4), (ii) plotting detected signal(y-axis) against log-transformed concentrations of binding protein orreference anti-PF-4 antibody (x-axis), and (iii) determining the areaunder the curve (AUC, or total peak area) from these non-curve fitteddata in the measured range (concentrations of binding protein orreference antibody from about 10 ng/ml to about 10000 ng/ml). Accordingto a particular embodiment of the invention, determining thecross-reaction to PF-4 by aligned sandwich-ELISA comprises thefollowing: the wells of a protein adsorbing microtiter plate are coatedwith a certain amount of an aligning antibody suitable to capture thebinding protein under investigation and the reference anti-PF-4antibody, for example 100 μl/well of 50 μg/ml Fc specific anti-mouseIgG, Sigma cat. no.: M3534, in 100 mM sodium hydrogen carbonate, pH9.6); the plate is then washed, blocked, and washed again; thencontacted with a ˜1:3 dilution series of the binding protein underinvestigation or of the reference anti-PF-4 antibody from about 10 ng/mlto about 10000 ng/ml (final concentration); after another washing stepthe plate is contacted with, e.g. 1:10 diluted, human or cynomolgusplasma, e.g. human plasma spiked with human PF-4, followed by detectionof the PF-4 bound to the plate, e.g. by means of a primary PF-4 specificantibody, an enzyme-conjugated secondary antibody and a colorimetricreaction.

According to one aspect of the invention, the cross-reaction of Aβbinding protein of the present invention to PF-4, when analyzed viasandwich-ELISA with cynomolgus plasma as described herein, is smallerthan the corresponding cross-reaction of a reference anti-PF-4 antibody,for example at least 2 times, at least 5 times, at least 10 times, atleast 20 times, or at least 30 times smaller; and/or, when analyzed viasandwich-ELISA with human plasma as described herein, is smaller thanthe corresponding cross-reaction of a reference anti-PF-4 antibody, forexample or at least 2 times, at least 5 times, at least 10 times, atleast 15 times, or at least 20 times smaller.

According to another aspect of the invention, the cross-reaction of Aβbinding protein of the present invention to PF-4, when analyzed viaaligned sandwich-ELISA with cynomolgus plasma as described herein, issmaller than the corresponding cross-reaction of a reference anti-PF-4antibody, for example at least 2 times, at least 5 times, at least 10times, at least 20 times, at least 30 times, at least 50 times, at least80 times or at least 115 times smaller; and/or, when analyzed viaaligned sandwich-ELISA with human plasma as described herein, is smallerthan the corresponding cross-reaction of a reference anti-PF-4 antibody,for example at least 2 times, at least 5 times, at least 10 times, atleast 15 times, at least 20 times, at least 25 times smaller.

According to another aspect of the invention, the cross-reaction of Aβbinding protein of the present invention to PF-4, when analyzed viasandwich-ELISA and aligned sandwich-ELISA with cynomolgus plasma asdescribed herein, is smaller than the corresponding cross-reaction of areference anti-PF-4 antibody, for example at least 2 times, at least 5times, at least 10 times, at least 20 times, or at least 30 timessmaller.

According to another aspect of the invention, the cross-reaction of Aβbinding protein of the present invention to PF-4, when analyzed viasandwich-ELISA and aligned sandwich-ELISA with human plasma as describedherein, is smaller than the corresponding cross-reaction of a referenceanti-PF-4 antibody, for example at least 2 times, at least 5 times, atleast 10 times, at least 20 times, or at least 30 times smaller.

According to another aspect of the invention, the cross-reaction of Aβbinding protein of the present invention to PF-4, when analyzed viasandwich-ELISA and aligned sandwich-ELISA with cynomolgus and humanplasma as described herein, is smaller than the correspondingcross-reaction of a reference anti-PF-4 antibody, for example at least 2times, at least 5 times, at least 10 times, at least 20 times, or atleast 30 times smaller.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering”, as used herein, refers to the process ofrendering a chemical species such as a polypeptide substantially free ofnaturally associated components by isolation, e.g., using proteinpurification techniques well known in the art.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, mean that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such functional fragment,mutant, variant, or derivative antibody formats are known in the art.Nonlimiting embodiments of which are discussed below. A “full-lengthantibody”, as used herein, refers to an Ig molecule comprising fourpolypeptide chains, two heavy chains and two light chains. The chainsare usually linked to one another via disulfide bonds. Each heavy chainis comprised of a heavy chain variable region (also referred to hereinas “variable heavy chain”, or abbreviated herein as HCVR or VH) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (also referred to herein as“variable light chain”, or abbreviated herein as LCVR or VL) and a lightchain constant region. The light chain constant region is comprised ofone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 Immunoglobulinmolecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG 1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The terms “antigen-binding portion” of an antibody (or simply “antibodyportion”), “antigen-binding moiety” of an antibody (or simply “antibodymoiety”), as used herein, refers to one or more fragments of an antibodythat retain the ability to specifically bind to an antigen (e.g.,Aβ(20-42) globulomer), i.e. are functional fragments of an antibody. Ithas been shown that the antigen-binding function of an antibody can beperformed by one or more fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific, specifically binding to two or more different antigens.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., Nature 341: 544-546, 1989; Winter et al., WO 90/05144 A1,herein incorporated by reference), which comprises a single variabledomain; and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al.,Science 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci.USA 85: 5879-5883, 1988). Such single chain antibodies are alsoencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies, are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993;Poljak et al., Structure 2: 1121-1123, 1994). Such antibody bindingportions are known in the art (Kontermann and Dubel eds., AntibodyEngineering, Springer-Verlag. New York. 790 pp., 2001, ISBN3-540-41354-5).

The term “antibody”, as used herein, also comprises antibody constructs.The term “antibody construct” as used herein refers to a polypeptidecomprising one or more of the antigen-binding portions of the inventionlinked to a linker polypeptide or an immunoglobulin constant domain.Linker polypeptides comprise two or more amino acid residues joined bypeptide bonds and are used to link one or more antigen binding portions.Such linker polypeptides are well known in the art (see e.g., Holligeret al., Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993; Poljak et al.,Structure 2: 1121-1123, 1994).

An immunoglobulin constant domain refers to a heavy or light chainconstant domain. Human IgG heavy chain and light chain constant domainamino acid sequences are known in the art and represented in Table 1.

TABLE 1 SEQUENCE OF HUMAN IgG HEAVY CHAIN CONSTANT DOMAIN AND LIGHTCHAIN CONSTANT DOMAIN Sequence Protein Identifier Sequence123456789012345678901234567890 Ig gamma-1 constant SEQ ID NO: 25ASTKGPSVFFLAPSSKSTSGGTAALGCLVK region DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig gamma-1 constant SEQ ID NO: 26ASTKGPSVFPLAPSSKSTSGGTAALGCLVK region mutantDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Ig Kappaconstant SEQ ID NO: 27 TVAAPSVFIFPPSDEQLKSGTASVVCLLNN regionFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Ig Lambda constant SEQ ID NO: 28QPKAAPSVTLFPPSSEELQANKATLVCLIS region DFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS

Still further, a binding protein of the present invention (e.g. anantibody) may be part of a larger immunoadhesion molecule, formed bycovalent or noncovalent association of the binding protein of theinvention with one or more other proteins or peptides. Examples of suchimmunoadhesion molecules include the use of the streptavidin core regionto make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodiesand Hybridomas 6: 93-101, 1995) and use of a cysteine residue, a markerpeptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058, 1994). Antibody portions, such as Fab and F(ab′)₂ fragments,can be prepared from whole antibodies using conventional techniques,such as papain or pepsin digestion, respectively, of whole antibodies.Moreover, antibodies, antibody portions and immunoadhesion molecules canbe obtained using standard recombinant DNA techniques, as describedherein.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities. An isolated antibody that specifically bindsAβ(20-42) globulomer may, however, have cross-reactivity to otherantigens, such as Aβ globulomers, e.g. Aβ(12-42) globulomer or other Aβforms. Moreover, an isolated antibody may be substantially free of othercellular material and/or chemicals and/or any other targeted Aβ form.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g. mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular in CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther in Section B, below), antibodies isolated from a recombinant,combinatorial human antibody library (Hoogenboom, TIB Tech. 15: 62-70,1997; Azzazy and Highsmith, Clin. Biochem. 35: 425-445, 2002; GavilondoJ. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H.,and Chames P. (2000) Immunology Today 21:371-378), antibodies isolatedfrom an animal (e.g. a mouse) that is transgenic for humanimmunoglobulin genes (see e.g. Taylor, L. D., et al. (1992) Nucl. AcidsRes. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) CurrentOpinion in Biotechnology 13:593-597; Little M. et al (2000) ImmunologyToday 21:364-370) or antibodies prepared, expressed, created or isolatedby any other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine CDRs (e.g., CDR3) in which one or more of the murinevariable heavy and light chain regions has been replaced with humanvariable heavy and light chain sequences.

The terms “Kabat numbering”, “Kabat definitions and “Kabat labeling” areused interchangeably herein. These terms, which are recognized in theart, refer to a system of numbering amino acid residues which are morevariable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). For the heavy chainvariable region, the hypervariable region ranges from amino acidpositions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, andamino acid positions 95 to 102 for CDR3. For the light chain variableregion, the hypervariable region ranges from amino acid positions 24 to34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acidpositions 89 to 97 for CDR3.

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, for exampleat least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,despite having great diversity at the level of amino acid sequence.These sub-portions were designated as L1, L2 and L3 or H1, H2 and H3where the “L” and the “H” designates the light chain and the heavychains regions, respectively. These regions may be referred to asChothia CDRs, which have boundaries that overlap with Kabat CDRs. Otherboundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems, particular embodiments use Kabat orChothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In one embodiment, the donorantibody is an antibody from a species different from the antibody fromwhich the framework regions are obtained or derived. In the context of ahumanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain acceptor sequences are known in theart. In one embodiment of the invention, the human heavy chain and lightchain acceptor sequences are selected from the sequences described inTable 2 and Table 3. In another embodiment, the human heavy chain andlight chain acceptor sequences are selected from sequences which are atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequences described in Table 2 and Table 3.

TABLE 2 HEAVY CHAIN ACCEPTOR SEQUENCES SEQ ID NO Protein region Sequence123456789012345678901234567890 34 VH3_53/JH6 FR1EVQLVESGGGLIQPGGSLRLSCAASGFTVS 35 VH3_53/JH6 FR2 WVRQAPGKGLEWVS 36VH3_53/JH6 FR3 RFTISRDNSKNTLYLQMNSLRAEDTAVYYC AR 37 VH3_53/JH6 FR4WGQGTTVTVSS 38 VH4_59/JH6 FR1 QVQLQESGPGLVKPSETLSLTCTVSGGSIS 39VH4_59/JH6 FR2 WIRQPPGKGLEWIG 40 VH4_59/JH6 FR3RVTISVDTSKNQFSLKLSSVTAADTAVYYC AR 41 VH4_59/JH6 FR4 WGQGTTVTVSS

TABLE 3 LIGHT CHAIN ACCEPTOR SEQUENCES SEQ ID NO Protein region Sequence123456789012345678901234567890 42 A1/2-30/Jκ2 FR1DVVMTQSPLSLPVTLGQPASISC 43 A1/2-30/Jκ2 FR2 WFQQRPGQSPRRLIY 44A1/2-30/Jκ2 FR3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVY YC 45 A1/2-30/Jκ2 FR4FGQGTKLEIKR

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)).One of the advantages provided by various embodiments of the presentinvention stems from the recognition that germline antibody genes aremore likely than mature antibody genes to conserve essential amino acidsequence structures characteristic of individuals in the species, henceless likely to be recognized as from a foreign source when usedtherapeutically in that species.

As used herein, the term “key” residues refer to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” is an antibody or avariant, derivative, analog or portion thereof which immunospecificallybinds to an antigen of interest and which comprises a framework (FR)region having substantially the amino acid sequence of a human antibodyand a complementary determining region (CDR) having substantially theamino acid sequence of a non-human antibody. As used herein, the term“substantially” in the context of a CDR refers to a CDR having an aminoacid sequence at least 90%, at least 95%, at least 98% or at least 99%identical to the amino acid sequence of a non-human antibody CDR. Ahumanized antibody comprises substantially all of at least one, andtypically two, variable domains (Fab, Fab′, F(ab′)₂, FabC, Fv) in whichall or substantially all of the CDR regions correspond to those of anon-human immunoglobulin (i.e., donor antibody) and all or substantiallyall of the framework regions are those of a human immunoglobulinconsensus sequence. According to one aspect, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. In some embodiments, ahumanized antibody contains both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include the CH1,hinge, CH2, CH3, and CH4 regions of the heavy chain. In someembodiments, a humanized antibody only contains a humanized light chain.In some embodiments, a humanized antibody only contains a humanizedheavy chain. In specific embodiments, a humanized antibody only containsa humanized variable domain of a light chain and/or of a heavy chain.

The humanized antibody can be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including without limitation IgG 1, IgG2, IgG3 and IgG4. The humanizedantibody may comprise sequences from more than one class or isotype, andparticular constant domains may be selected to optimize desired effectorfunctions using techniques well-known in the art.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion and/or deletion of at least one amino acid residue so that theCDR or framework residue at that site does not correspond to either thedonor antibody or the consensus framework. In one embodiment, suchmutations, however, will not be extensive. Usually, at least 90%, atleast 95%, at least 98%, or at least 99% of the humanized antibodyresidues will correspond to those of the parental FR and CDR sequences.As used herein, the term “consensus framework” refers to the frameworkregion in the consensus immunoglobulin sequence. As used herein, theterm “consensus immunoglobulin sequence” refers to the sequence formedfrom the most frequently occurring amino acids (or nucleotides) in afamily of related immunoglobulin sequences (See e.g., Winnaker, FromGenes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In afamily of immunoglobulins, each position in the consensus sequence isoccupied by the amino acid occurring most frequently at that position inthe family. If two amino acids occur equally frequently, either can beincluded in the consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “antibody”, as used herein, also comprises multivalent bindingproteins. The term “multivalent binding protein” is used in thisspecification to denote a binding protein comprising two or more antigenbinding sites. The multivalent binding protein is engineered to have thethree or more antigen binding sites, and is generally not a naturallyoccurring antibody. The term “multispecific binding protein” refers to abinding protein capable of binding two or more related or unrelatedtargets. Dual variable domain (DVD) binding proteins as used herein, arebinding proteins that comprise two or more antigen binding sites and aretetravalent or multivalent binding proteins. Such DVDs may bemonospecific, i.e. capable of binding one antigen or multispecific, i.e.capable of binding two or more antigens. DVD binding proteins comprisingtwo heavy chain DVD polypeptides and two light chain DVD polypeptidesare referred to a DVD Ig. Each half of a DVD Ig comprises a heavy chainDVD polypeptide, and a light chain DVD polypeptide, and two antigenbinding sites. Each binding site comprises a heavy chain variable domainand a light chain variable domain with a total of 6 CDRs involved inantigen binding per antigen binding site. DVD binding proteins andmethods of making DVD binding proteins are disclosed in U.S. patentapplication Ser. No. 11/507,050 and incorporated herein by reference.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound by abinding protein, in particular by an antibody. In certain embodiments, abinding protein or an antibody is said to specifically bind an antigenwhen it preferentially recognizes its target antigen in a complexmixture of proteins and/or macromolecules.

The binding affinities of the antibodies of the invention may beevaluated by using standardized in-vitro immunoassays such as ELISA, dotblot or BIAcore analyses (Pharmacia Biosensor AB, Uppsala, Sweden andPiscataway, N.J.). For further descriptions, see Jönsson, U., et al.(1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991)Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

According to a particular embodiment, the affinities defined hereinrefer to the values obtained by performing a dot blot and evaluating itby densitometry. According to a particular embodiment of the invention,determining the binding affinity by dot blot comprises the following: acertain amount of the antigen (e.g. the Aβ(X-Y) globulomer, Aβ(X-Y)monomer or Aβ(X-Y) fibrils, as defined above) or, expediently, anappropriate dilution thereof, for instance in 20 mM NaH₂PO₄, 140 mMNaCl, pH 7.4, 0.2 mg/ml BSA to an antigen concentration of, for example,100 pmol/μl, 10 pmol/μl, 1 pmol/μl, 0.1 pmol/μl and 0.01 pmol/μl, isdotted onto a nitrocellulose membrane, the membrane is then blocked withmilk to prevent unspecific binding and washed, then contacted with theantibody of interest followed by detection of the latter by means of anenzyme-conjugated secondary antibody and a colorimetric reaction; atdefined antibody concentrations, the amount of antibody bound allowsaffinity determination. Thus the relative affinity of two differentantibodies to one target, or of one antibody to two different targets,is here defined as the relation of the respective amounts oftarget-bound antibody observed with the two antibody-target combinationsunder otherwise identical dot blot conditions. Unlike a similar approachbased on Western blotting, the dot blot approach will determine anantibody's affinity to a given target in the latter's naturalconformation; unlike the ELISA approach, the dot blot approach does notsuffer from differences in the affinities between different targets andthe matrix, thereby allowing for more precise comparisons betweendifferent targets.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jönsson, U., et al. (1993) Ann. Biol. Clin.,51: 19-26; Jönsson et al., (1991) BioTechniques, 11: 620-627; Johnssonet al., (1995) J. Mol. Recognit., 8: 125-131; and Johnnson et al. (1991)Anal. Biochem., 198: 268-277.

The term “k_(on)” (also “Kon”, “kon”, “K_(on)”), as used herein, isintended to refer to the on-rate constant for association of a bindingprotein (e.g., an antibody) to an antigen to form an associationcomplex, e.g., antibody/antigen complex, as is known in the art. The“k.” also is known by the terms “association rate constant”, or “ka”, asused interchangeably herein. This value indicates the binding rate of abinding protein (e.g., an antibody) to its target antigen or the rate ofcomplex formation between a binding protein (e.g., an antibody) andantigen as is shown by the equation below:Antibody (“Ab”)+Antigen (“Ag”)→Ab-Ag.

The term “k_(off)” (also “Koff”, “koff”, “K_(off)”), as used herein, isintended to refer to the off rate constant for dissociation, or“dissociation rate constant”, of a binding protein (e.g., an antibody)from an association complex (e.g., an antibody/antigen complex) as isknown in the art. This value indicates the dissociation rate of abinding protein (e.g., an antibody) from its target antigen, orseparation of the Ab-Ag complex over time into free antibody and antigenas shown by the equation below:Ab+Ag←Ab-Ag.

The term “K_(D)” (also “K_(d)” or “KD”), as used herein, is intended torefer to the “equilibrium dissociation constant”, and refers to thevalue obtained in a titration measurement at equilibrium, or by dividingthe dissociation rate constant (k_(off)) by the association rateconstant (k_(on)). The association rate constant (k_(on)), thedissociation rate constant (k_(off)), and the equilibrium dissociationconstant (K_(D)) are used to represent the binding affinity of a bindingprotein (e.g., an antibody) to an antigen. Methods for determiningassociation and dissociation rate constants are well known in the art.Using fluorescence-based techniques offers high sensitivity and theability to examine samples in physiological buffers at equilibrium.Other experimental approaches and instruments such as a BIAcore®(biomolecular interaction analysis) assay can be used (e.g., instrumentavailable from BIAcore International AB, a GE Healthcare company,Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay)assay, available from Sapidyne Instruments (Boise, Id.) can also beused.

The term “labeled binding protein”, as used herein, refers to a bindingprotein with a label incorporated that provides for the identificationof the binding protein. Likewise, the term “labeled antibody” as usedherein, refers to an antibody with a label incorporated that providesfor the identification of the antibody. In one aspect, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). Examples of labels for polypeptides include, but are notlimited to, the following: radioisotopes or radionuclides (e.g., ³H,¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm);fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),enzymatic labels (e.g., horseradish peroxidase, luciferase, alkalinephosphatase); chemiluminescent markers; biotinyl groups; predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags); and magnetic agents, such as gadoliniumchelates.

The term “antibody”, as used herein, also comprises antibody conjugates.The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic agent.

The term “therapeutic agent” is used herein to denote a chemicalcompound, a mixture of chemical compounds, a biological macromolecule,or an extract made from biological materials that is a “cognitiveenhancing drug,” which is a drug that improves impaired human cognitiveabilities of the brain (namely, thinking, learning, and memory).Cognitive enhancing drugs work by altering the availability ofneurochemicals (e.g., neurotransmitters, enzymes, and hormones), byimproving oxygen supply, by stimulating nerve growth, or by inhibitingnerve damage. Examples of cognitive enhancing drugs include a compoundthat increases the activity of acetylcholine such as, but not limitedto, an acetylcholine receptor agonist (e.g., a nicotinic α-7 receptoragonist or allosteric modulator, an α4β2 nicotinic receptor agonist orallosteric modulators), an acetylcholinesterase inhibitor (e.g.,donepezil, rivastigmine, and galantamine), a butyrylcholinesteraseinhibitor, an N-methyl-D-aspartate (NMDA) receptor antagonist (e.g.,memantine), an activity-dependent neuroprotective protein (ADNP)agonist, a serotonin 5-HT1A receptor agonist (e.g., xaliproden), a 5-HT₄receptor agonist, a 5-HT₆ receptor antagonist, a serotonin 1A receptorantagonist, a histamine H₃ receptor antagonist, a calpain inhibitor, avascular endothelial growth factor (VEGF) protein or agonist, a trophicgrowth factor, an anti-apoptotic compound, an AMPA-type glutamatereceptor activator, a L-type or N-type calcium channel blocker ormodulator, a potassium channel blocker, a hypoxia inducible factor (HIF)activator, a HIF prolyl 4-hydroxylase inhibitor, an anti-inflammatoryagent, an inhibitor of amyloid Aβ peptide or amyloid plaque, aninhibitor of tau hyperphosphorylation, a phosphodiesterase 5 inhibitor(e.g., tadalafil, sildenafil), a phosphodiesterase 4 inhibitor, amonoamine oxidase inhibitor, or pharmaceutically acceptable saltthereof. Specific examples of such cognitive enhancing drugs include,but are not limited to, cholinesterase inhibitors such as donepezil(Aricept®), rivastigmine (Exelon®), galanthamine (Reminyl®),N-methyl-D-aspartate antagonists such as memantine (Namenda®).

The terms “crystal” and “crystallized”, as used herein, refer to abinding protein (e.g., an antibody, or antigen binding portion thereof),that exists in the form of a crystal. Crystals are one form of the solidstate of matter, which is distinct from other forms such as theamorphous solid state or the liquid crystalline state. Crystals arecomposed of regular, repeating, three-dimensional arrays of atoms, ions,molecules (e.g., proteins such as antibodies), or molecular assemblies(e.g., antigen/antibody complexes). These three-dimensional arrays arearranged according to specific mathematical relationships that arewell-understood in the field. The fundamental unit, or building block,that is repeated in a crystal is called the asymmetric unit. Repetitionof the asymmetric unit in an arrangement that conforms to a given,well-defined crystallographic symmetry provides the “unit cell” of thecrystal. Repetition of the unit cell by regular translations in allthree dimensions provides the crystal. See Giege, R. and Ducruix, A.Barrett, Crystallization of Nucleic Acids and Proteins, a PracticalApproach, 2^(nd) ed., pp. 20 1-16, Oxford University Press, New York,N.Y., (1999).”

As used herein, the term “neutralizing” refers to neutralization ofbiological activity of a targeted Aβ form when a binding proteinspecifically binds said Aβ form. For example, a neutralizing bindingprotein is a neutralizing antibody whose binding to the Aβ(20-42) aminoacid region of the globulomer (and/or any other targeted Aβ form)results in inhibition of a biological activity of the globulomer.According to one aspect of the invention, the neutralizing bindingprotein binds to the Aβ(20-42) region of the globulomer (and/or anyother targeted Aβ form), and reduces a biologically activity of thetargeted Aβ form by at least about 20%, 40%, 60%, 80%, 85% or moreInhibition of a biological activity of the targeted Aβ form by aneutralizing binding protein can be assessed by measuring one or moreindicators of the targeted Aβ form biological activity well known in theart, for example interaction (e.g. binding) of the targeted Aβ form to aP/Q type voltage-gated presynaptic calcium channel, inhibition of P/Qtype voltage-gated presynaptic calcium channel activity, Ca⁺⁺ fluxthrough P/Q type voltage-gated presynaptic calcium channel, local (e.g.intracellular) Ca⁺⁺ concentration, synaptic activity.

The term “activity” includes activities such as the bindingspecificity/affinity of a binding protein, in particular of an antibody,for an antigen, for example an Aβ(20-42) globulomer (and any othertargeted Aβ form); and/or the neutralizing potency of an antibody, forexample an antibody whose binding to a targeted Aβ form inhibits thebiological activity of the targeted Aβ form. Said biological activity ofthe targeted Aβ form comprises interaction of the Aβ form to P/Q typevoltage-gated presynaptic calcium channels, which results in inhibitionof the activity of said calcium channels.

The subject invention also provides isolated nucleotide sequencesencoding the binding proteins of the present invention. The presentinvention also provides those nucleotide sequences (or fragmentsthereof) having sequences comprising, corresponding to, identical to,hybridizable to, or complementary to, at least about 70% (e.g., 70% 71%,72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%), at least about 80% (e.g.,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%), or at least about90% (e.g, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identityto these encoding nucleotide sequences. (All integers (and portionsthereof) between and including 70% and 100% are considered to be withinthe scope of the present invention with respect to percent identity.)Such sequences may be derived from any source (e.g., either isolatedfrom a natural source, produced via a semi-synthetic route, orsynthesized de novo). In particular, such sequences may be isolated orderived from sources other than described in the examples (e.g.,bacteria, fungus, algae, mouse or human).

For purposes of the present invention, a “fragment” of a nucleotidesequence is defined as a contiguous sequence of approximately at least6, e.g. at least about 8, at least about 10 nucleotides, or at leastabout 15 nucleotides, corresponding to a region of the specifiednucleotide sequence.

The term “identity” refers to the relatedness of two sequences on anucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments (or two amino acid sequences).“Percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over a particular region, determining thenumber of positions at which the identical base or amino acid occurs inboth sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the segment being compared and multiplying the result by 100. Optimalalignment of sequences may be conducted by the algorithm of Smith &Waterman, Appl. Math. 2: 482, 1981, by the algorithm of Needleman &Wunsch, J. Mol. Biol. 48: 443, 1970, by the method of Pearson & Lipman,Proc. Natl. Acad. Sci. (USA) 85: 2444, 1988, and by computer programswhich implement the relevant algorithms (e.g., Clustal Macaw Pileup(Higgins et al., CABIOS. 5L151-153, 1989), FASTDB (Intelligenetics),BLAST (National Center for Biomedical Information; Altschul et al.,Nucleic Acids Research 25: 3389-3402, 1997), PILEUP (Genetics ComputerGroup, Madison, Wis.) or GAP, BESTFIT, FASTA and TFASTA (WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, Madison,Wis.)). (See U.S. Pat. No. 5,912,120.)

For purposes of the present invention, “complementarity” is defined asthe degree of relatedness between two DNA segments. It is determined bymeasuring the ability of the sense strand of one DNA segment tohybridize with the anti-sense strand of the other DNA segment, underappropriate conditions, to form a double helix. A “complement” isdefined as a sequence which pairs to a given sequence based upon thecanonic base-pairing rules. For example, a sequence A-G-T in onenucleotide strand is “complementary” to T-C-A in the other strand. Inthe double helix, adenine appears in one strand, thymine appears in theother strand. Similarly, wherever guanine is found in one strand,cytosine is found in the other. The greater the relatedness between thenucleotide sequences of two DNA segments, the greater the ability toform hybrid duplexes between the strands of the two DNA segments.

“Similarity” between two amino acid sequences is defined as the presenceof a series of identical as well as conserved amino acid residues inboth sequences. The higher the degree of similarity between two aminoacid sequences, the higher the correspondence, sameness or equivalenceof the two sequences. (“Identity between two amino acid sequences isdefined as the presence of a series of exactly alike or invariant aminoacid residues in both sequences.) The definitions of “complementarity”,“identity” and “similarity” are well known to those of ordinary skill inthe art.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 amino acids, e.g.at least 8 amino acids, or at least 15 amino acids, from a polypeptideencoded by the nucleic acid sequence.

The term “polynucleotide” as referred to herein, means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA, but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or somecombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide”: is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, to the progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein. In oneaspect, host cells include prokaryotic and eukaryotic cells selectedfrom any of the kingdoms of life. Eukaryotic cells include protist,fungal, plant and animal cells. In another aspect host cells include,but are not limited to, the prokaryotic cell line E. coli; mammaliancell lines CHO, HEK 293 and COS; the insect cell line Sf9; and thefungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al. Molecular Cloning: ALaboratory Manual (2^(nd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct, which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

The terms “regulate” and “modulate” are used interchangeably, and, asused herein, refer to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of a targeted Aβform). Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of a targeted Aβ form). Forexample, a modulator may cause an increase or decrease in the magnitudeof a certain activity or function of a molecule compared to themagnitude of the activity or function observed in the absence of themodulator. In certain embodiments, a modulator is an inhibitor, whichdecreases the magnitude of at least one activity or function of amolecule.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist.

The term “antagonist” or “inhibitor”, as used herein, refer to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological activity of atargeted Aβ form. Antagonists and inhibitors of a targeted Aβ form mayinclude, but are not limited to, the binding proteins of the invention,which bind to Aβ(20-42) globulomer and any other targeted Aβ form. Anantagonist or inhibitor of a targeted Aβ form may, for example, reducethe inhibitory effect of said AP form on the activity of a P/Q typevoltage-gated presynaptic calcium channel.

As used herein, the term “effective amount” refers to the amount of atherapy which is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof, prevent theadvancement of a disorder, cause regression of a disorder, prevent therecurrence, development, onset or progression of one or more symptomsassociated with a disorder, detect a disorder, or enhance or improve theprophylactic or therapeutic effect(s) of another therapy (e.g.,prophylactic or therapeutic agent).

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, serum, urine, synovial fluid, cells,organs, tissues, bone marrow, lymph nodes and spleen.

I. Antibodies of the Invention

A first particular aspect of the invention provides CDR graftedantibodies, or antigen-binding portions thereof, that bind Aβ(20-42)globulomer and/or any other targeted Aβ form. A second particular aspectof the invention provides humanized antibodies, or antigen-bindingportions thereof, that bind Aβ(20-42) globulomer and/or any othertargeted Aβ form. According to one particular aspect, the antibodies, orportions thereof, are isolated antibodies. According to a furtherparticular aspect, the antibodies of the invention neutralize anactivity of Aβ(20-42) globulomer and/or of any other targeted Aβ form.

A. Method of Making Anti-Aβ(20-42) Globulomer Antibodies

Antibodies of the present invention may be made by any of a number oftechniques known in the art.

1. Anti-Aβ(20-42) Globulomer Monoclonal Antibodies Using HybridomaTechnology

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In oneembodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, e.g., the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention. Briefly, mice can be immunized withan Aβ(20-42) globulomer antigen. In a particular embodiment, the antigenis administered with a adjuvant to stimulate the immune response. Suchadjuvants include complete or incomplete Freund's adjuvant, RIBI(muramyl dipeptides) or ISCOM (immunostimulating complexes). Suchadjuvants may protect the polypeptide from rapid dispersal bysequestering it in a local deposit, or they may contain substances thatstimulate the host to secrete factors that are chemotactic formacrophages and other components of the immune system. Preferably, if apolypeptide is being administered, the immunization schedule willinvolve two or more administrations of the polypeptide, spread out overseveral weeks.

After immunization of an animal with an Aβ(20-42) globulomer antigen,antibodies and/or antibody-producing cells may be obtained from theanimal. An anti-Aβ(20-42) globulomer antibody-containing serum isobtained from the animal by bleeding or sacrificing the animal. Theserum may be used as it is obtained from the animal, an immunoglobulinfraction may be obtained from the serum, or the anti-Aβ(20-42)globulomer antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen Aβ(20-42) globulomer are detected in the mouse serum, the mousespleen is harvested and splenocytes isolated. The splenocytes are thenfused by well-known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding Aβ(20-42) globulomer. Ascites fluid, whichgenerally contains high levels of antibodies, can be generated byimmunizing mice with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art (See, e.g., Harlow and Lane, supra).In a particular embodiment, the myeloma cells do not secreteimmunoglobulin polypeptides (a non-secretory cell line). After fusionand antibiotic selection, the hybridomas are screened using Aβ(20-42)globulomer, or a portion thereof, or a cell expressing Aβ(20-42)globulomer. In a particular embodiment, the initial screening isperformed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay (RIA). An example of ELISA screening is provided in WO00/37504, herein incorporated by reference.

Anti-Aβ(20-42) globulomer antibody-producing hybridomas are selected,cloned and further screened for desirable characteristics, includingrobust hybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

In a particular embodiment, the hybridomas are mouse hybridomas, asdescribed above. In another particular embodiment, the hybridomas areproduced in a non-human, non-mouse species such as rats, sheep, pigs,goats, cattle or horses. In another embodiment, the hybridomas are humanhybridomas, in which a human non-secretory myeloma is fused with a humancell expressing an anti-Aβ(20-42) globulomer antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

2. Anti-Aβ(20-42) Globulomer Monoclonal Antibodies Using Slam

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052, PCT Publication WO92/02551 and Babcock, J. S.et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described in Section 1, arescreened using an antigen-specific hemolytic plaque assay, wherein theantigen Aβ(20-42) globulomer, or a subunit thereof, is coupled to sheepred blood cells using a linker, such as biotin, and used to identifysingle cells that secrete antibodies with specificity for Aβ(20-42)globulomer. Following identification of antibody-secreting cells ofinterest, heavy- and light-chain variable region cDNAs are rescued fromthe cells by reverse transcriptase-PCR and these variable regions canthen be expressed, in the context of appropriate immunoglobulin constantregions (e.g., human constant regions), in mammalian host cells, such asCOS or CHO cells. The host cells transfected with the amplifiedimmunoglobulin sequences, derived from in vivo selected lymphocytes, canthen undergo further analysis and selection in vitro, for example bypanning the transfected cells to isolate cells expressing antibodies toAβ(20-42) globulomer. The amplified immunoglobulin sequences further canbe manipulated in vitro, such as by in vitro affinity maturation methodssuch as those described in PCT Publication WO 97/29131 and PCTPublication WO 00/56772.

3. Anti-Aβ(20-42) Globulomer Monoclonal Antibodies Using TransgenicAnimals

In another embodiment of the instant invention, antibodies are producedby immunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with an Aβ(20-42) globulomer antigen. In aparticular embodiment, the non-human animal is a XENOMOUSE transgenicmouse, an engineered mouse strain that comprises large fragments of thehuman immunoglobulin loci and is deficient in mouse antibody production.See, e.g., Green et al. Nature Genetics 7:13-21 (1994) and U.S. Pat.Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001,6,114,598 and 6,130,364. See also WO 91/10741, published Jul. 25, 1991,WO 94/02602, published Feb. 3, 1994, WO 96/34096 and WO 96/33735, bothpublished Oct. 31, 1996, WO 98/16654, published Apr. 23, 1998, WO98/24893, published Jun. 11, 1998, WO 98/50433, published Nov. 12, 1998,WO 99/45031, published Sep. 10, 1999, WO 99/53049, published Oct. 21,1999, WO 00 09560, published Feb. 24, 2000 and WO 00/037504, publishedJun. 29, 2000. The XENOMOUSE transgenic mouse produces an adult-likehuman repertoire of fully human antibodies, and generatesantigen-specific human monoclonal antibodies. The XENOMOUSE transgenicmouse contains approximately 80% of the human antibody repertoirethrough introduction of megabase sized, germline configuration YACfragments of the human heavy chain loci and x light chain loci. SeeMendez et al., Nature Genetics 15:146-156 (1997), Green and JakobovitsJ. Exp. Med. 188:483-495 (1998), the disclosures of which are herebyincorporated by reference.

4. Anti-Aβ(20-42) Globulomer Monoclonal Antibodies Using RecombinantAntibody Libraries

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT Publication No. WO92/18619; Dower et al.PCT Publication No. WO91/17271; Winter et al. PCT Publication No.WO92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling etal. PCT Publication No. WO93/01288; McCafferty et al. PCT PublicationNo. WO92/01047; Garrard et al. PCT Publication No. WO92/09690; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum AntibodHybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; McCaffertyet al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson etal. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS88:7978-7982, US patent application publication 20030186374, and PCTPublication No. WO97/29131, the contents of each of which areincorporated herein by reference.

The recombinant antibody library may be from a subject immunized withAβ(20-42) globulomer, or a portion of Aβ(20-42) globulomer.Alternatively, the recombinant antibody library may be from a naïvesubject, i.e., one who has not been immunized with Aβ(20-42) globulomer,such as a human antibody library from a human subject who has not beenimmunized with human Aβ(20-42) globulomer. Antibodies of the inventionare selected by screening the recombinant antibody library with thepeptide comprising human Aβ(20-42) globulomer to thereby select thoseantibodies that recognize Aβ(20-42) globulomer and discriminate Aβ(1-42)globulomer, Aβ(1-40) and Aβ(1-42) monomer, Aβ-fibrils and sAPPα. Methodsfor conducting such screening and selection are well known in the art,such as described in the references in the preceding paragraph. Toselect antibodies of the invention having particular binding affinitiesfor Aβ(20-42) globulomer and discriminate Aβ(1-42) globulomer, Aβ(1-40)and Aβ(1-42) monomer, Aβ-fibrils and sAPPα, such as those thatdissociate from human Aβ(20-42) globulomer with a particular koff rateconstant, the art-known method of dot blot can be used to selectantibodies having the desired koff rate constant. To select antibodiesof the invention having a particular neutralizing activity for Aβ(20-42)globulomer and discriminate Aβ(1-42) globulomer, Aβ(1-40) and Aβ(1-42)monomer, Aβ-fibrils and sAPPα, such as those with a particular an IC50standard methods known in the art for assessing the inhibition ofAβ(20-42) globulomer activity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human Aβ(20-42) globulomerand discriminates Aβ(1-42) globulomer, Aβ(1-40) and Aβ(1-42) monomer,Aβ-fibrils and sAPPα. According to one aspect, the antibody is aneutralizing antibody. In various embodiments, the antibody is arecombinant antibody or a monoclonal antibody.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO90/02809; WO91/10737; WO92/01047;WO92/18619; WO93/11236; WO95/15982; WO95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780, 225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258, 498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification of dualspecificity antibodies of the invention. One type of alternativeexpression system is one in which the recombinant antibody library isexpressed as RNA-protein fusions, as described in PCT Publication No. WO98/31700 by Szostak and Roberts, and in Roberts, R. W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this system, acovalent fusion is created between an mRNA and the peptide or proteinthat it encodes by in vitro translation of synthetic mRNAs that carrypuromycin, a peptidyl acceptor antibiotic, at their 3′ end. Thus, aspecific mRNA can be enriched from a complex mixture of mRNAs (e.g., acombinatorial library) based on the properties of the encoded peptide orprotein, e.g., antibody, or portion thereof, such as binding of theantibody, or portion thereof, to the dual specificity antigen. Nucleicacid sequences encoding antibodies, or portions thereof, recovered fromscreening of such libraries can be expressed by recombinant means asdescribed above (e.g., in mammalian host cells) and, moreover, can besubjected to further affinity maturation by either additional rounds ofscreening of mRNA-peptide fusions in which mutations have beenintroduced into the originally selected sequence(s), or by other methodsfor affinity maturation in vitro of recombinant antibodies, as describedabove.

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed Wittrup, etal. U.S. Pat. No. 6,699,658 incorporated herein by reference.

B. Production of Recombinant Aβ(20-42) Globulomer Antibodies

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.It is possible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells. According to a particular aspectof the invention, expression of antibodies is performed using eukaryoticcells, for example mammalian host cells, because such eukaryotic cells(and in particular mammalian cells) are more likely than prokaryoticcells to assemble and secrete a properly folded and immunologicallyactive antibody.

According to one aspect, mammalian host cells for expressing therecombinant antibodies of the invention include Chinese Hamster Ovary(CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin,(1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp(1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2cells. When recombinant expression vectors encoding antibodies genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibodies in the host cells or secretion of theantibodies into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention and theother heavy and light chain are specific for an antigen other than theantigens of interest by crosslinking an antibody of the invention to asecond antibody by standard chemical crosslinking methods.

In a particular system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

1. Anti-Aβ(20-42) Globulomer Murine Antibodies

Table 4 is a list of amino acid sequences of VH and VL regions of murine4D10.

TABLE 4 LIST OF AMINO ACID SEQUENCES OF VH AND VL REGIONS SEQ ID NOPROTEIN REGION SEQUENCE 123456789012345678901234567890 23 m4D10_VHQVQLKQSGPSLIQPSQSLSITCTVSGFSLT SYGVHWVRQSPGKGLEWLGVIWRGGRIDYNAAFMSRLSITKDNSKSQVFFKMNSLQADDT AIYYCARNSDVWGTGTTVTVSS 24 m4D10_VLDVVMTQTPLTLSVTIGQPASISCKSSQSLL DIDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGV YYCWQGTHFPYTFGGGTKLEIKR *CDRs areunderlined in murine light and heavy chains.

2. Anti-Aβ(20-42) Globulomer Chimeric Antibodies

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. Methods for producing chimericantibodies are known in the art and discussed in detail herein. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entireties. In addition, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454 which areincorporated herein by reference in their entireties) by splicing genesfrom a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used.

In one embodiment, the chimeric antibodies of the invention are producedby replacing the heavy chain constant region of the murine monoclonalanti-Aβ(20-42) globulomer antibody 4D10 described in WO2007/062852 witha human IgG1 constant region.

3. Anti-Aβ(20-42) Globulomer CDR Grafted Antibodies

CDR-grafted antibodies of the invention comprise heavy and light chainvariable region sequences from a human antibody wherein one or more ofthe CDR regions of VH and/or VL are replaced with CDR sequences of themurine antibodies of the invention. A framework sequence from any humanantibody may serve as the template for CDR grafting. However, straightchain replacement onto such a framework often leads to some loss ofbinding affinity to the antigen. The more homologous a human antibody isto the original murine antibody, the less likely the possibility thatcombining the murine CDRs with the human framework will introducedistortions in the CDRs that could reduce affinity. Therefore, the humanvariable framework chosen to replace the murine variable framework apartfrom the CDRs have for example at least a 65% sequence identity with themurine antibody variable region framework. The human and murine variableregions apart from the CDRs have for example at least 70%, least 75%sequence identity, or at least 80% sequence identity. Methods forproducing chimeric antibodies are known in the art and discussed indetail herein. (also see EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,352).

Table 5 below illustrates the sequences of CDR grafted antibodies of thepresent invention (4D10hum antibodies) and the CDRs contained therein.

TABLE 5 LIST OF AMINO ACID SEQUENCES OF VH AND VL REGIONS OF CDR GRAFTEDANTIBODIES SEQ ID NO PROTEIN REGION SEQUENCE123456789012345678901234567890 4 4D10hum_VH.1zEVQLVESGGGLIQPGGSLRLSCAASGFTVS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYNAAFMSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 8 4D10hum_VH.2zQVQLQESGPGLVKPSETLSLTCTVSGGSIS SYGVHWIRQPPGKGLEWIGVIWRGGRIDYNAAFMSRVTISVDTSKNQFSLKLSSVTAADT AVYYCARNSDVWGQGTTVTVSS 17 VH 4D10humResidues 31-35 of SEQ SYGVH CDR-H1 ID NOs: 4, 8 18 VH 4D10hum Residues50-65 of SEQ VIWRGGRIDYNAAFMS CDR-H2 ID NOs: 4, 8 19 VH 4D10hum Residues98-101 of NSDV CDR-H3 SEQ ID NOs: 4, 8 12 4D10hum_Vκ.1zDVVMTQSPLSLPVTLGQPASISCKSSQSLL DIDGKTYLNWFQQRPGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHFPYTFGQGTKLEIKR 20 VL 4D10humResidues 24-39 of SEQ KSSQSLLDIDGKTYLN CDR-L1 ID NO: 12 21 VL 4D10humResidues 55-61 of SEQ LVSKLDS CDR-L2 ID NO: 12 22 VL 4D10hum Residues94-102 of WQGTHFPYT CDR-L3 SEQ ID NO: 12 *CDRs are underlined inhumanized light and heavy chains.

4. Anti-Aβ(20-42) Globulomer Humanized Antibodies

Humanized antibodies are antibody molecules from non-human speciesantibody that binds the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule.

Known human Ig sequences are disclosed, e.g., Kabat et al., Sequences ofProteins of Immunological Interest, U.S. Dept. Health (1983), eachentirely incorporated herein by reference. Such imported sequences canbe used to reduce immunogenicity or reduce, enhance or modify binding,affinity, on-rate, off-rate, avidity, specificity, half-life, or anyother suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.Antibodies can be humanized using a variety of techniques known in theart, such as but not limited to those described in Jones et al., Nature321:522 (1986); Verhoeyen et al., Science 239:1534 (1988), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCTpublication WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630,US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP 239,400,U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483,5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352, 6,204,023,6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567, eachentirely incorporated herein by reference, included references citedtherein.

Table 6 below illustrates the sequences of humanized antibodies of thepresent invention (4D10hum antibodies) and the CDRs contained therein.

TABLE 6 LIST OF AMINO ACID SEQUENCES OF VH AND VL REGIONS OF HUMANIZEDANTIBODIES SEQ ID NO PROTEIN REGION SEQUENCE123456789012345678901234567890 5 4D10hum_VH.1EVQLVESGGGLVQPGGSLRLSCAASGFTVS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYNAAFMSRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 6 4D10hum_VH.1aEVQLVESGGGLVQPGGSLRLSCAVSGFTLS SYGVHWVRQAPGKGLEWLGVIWRGGRIDYNAAFMSRLTISKDNSKSTVYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 7 4D10hum_VH.1bEVQLVESGGGLIQPGGSLRLSCAASGFTLS SYGVHWVRQAPGKGLEWVSVIWRGGRIDYNAAFMSRFTISKDNSKNTLYLQMNSLRAEDT AVYYCARNSDVWGQGTTVTVSS 9 4D10hum_VH.2EVQLQESGPGLVKPSETLSLTCTVSGGSIS SYGVHWIRQPPGKGLEWIGVIWRGGRIDYNAAFMSRVTISVDTSKNQFSLKLSSVTAADT AVYYCARNSDVWGQGTTVTVSS 10 4D10hum_VH.2aEVQLQESGPGLVKPSETLSLTCTVSGFSLS SYGVHWVRQPPGKGLEWLGVIWRGGRIDYNAAFMSRLTISKDTSKSQVSLKLSSVTAADT AVYYCARNSDVWGQGTTVTVSS 11 4D10hum_VH.2bEVQLQESGPGLVKPSETLSLTCTVSGFSLS SYGVHWIRQPPGKGLEWIGVIWRGGRIDYNAAFMSRVTISKDTSKNQFSLKLSSVTAADT AVYYCARNSDVWGQGTTVTVSS 17 VH 4D10humResidues 31-35 of SEQ SYGVH CDR-H1 ID NOs: 5, 6, 7, 9, 10, 11 18 VH4D10hum Residues 50-65 of SEQ VIWRGGRIDYNAAFMS CDR-H2 ID NOs: 5, 6, 7,9, 10, 11 19 VH 4D10hum Residues 98-101 of NSDV CDR-H3 SEQ ID NOs: 5, 6,7, 9, 10, 11 13 4D10hum_Vκ.1 DVVMTQTPLSLPVTPGQPASISCKSSQSLLDIDGKTYLNWFLQKPGQSPQRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR 14 4D10hum_Vκ.1a DVVMTQTPLSLPVTPGQPASISCKSSQSLLDIDGKTYLNWLLQKPGQSPQRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR 15 4D10hum_Vκ.1b DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYLNWLLQRPGQSPRRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR 16 4D10hum_Vκ.1c DVVMTQTPLSLPVTLGQPASISCKSSQSLLDIDGKTYLNWFLQKPGQSPRRLIYLVSKLD SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPYTFGQGTKLEIKR 20 VL 4D10hum Residues 24-39 of SEQKSSQSLLDIDGKTYLN CDR-L1 ID NOs: 13, 14, 15, 16 21 VL 4D10hum Residues55-61 of SEQ LVSKLDS CDR-L2 ID NOs: 13, 14, 15, 16 22 VL 4D10humResidues 94-102 of WQGTHFPYT CDR-L3 SEQ ID NOs: 13, 14, 15, 16 *CDRs areunderlined in humanized light and heavy chains.

C. Antibodies and Antibody-Producing Cell Lines

According to one aspect, anti-Aβ(20-42) globulomer antibodies of thepresent invention or antibodies against any other targeted Aβ formexhibit a high capacity to reduce or to neutralize activity of Aβ(20-42)globulomer (and/or any other targeted Aβ form).

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantregion. According to one aspect, the heavy chain constant region is anIgG1 heavy chain constant region or an IgG4 heavy chain constant region.According to a further aspect, the antibody comprises a light chainconstant region, either a kappa light chain constant region or a lambdalight chain constant region. According to one aspect, the antibodycomprises a kappa light chain constant region. An antibody portion canbe, for example, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (Winter, et al. U.S. Pat. Nos.5,648,260 and 5,624,821). The Fc portion of an antibody mediates severalimportant effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled antibody wherein an antibody of theinvention is derivatized or linked to another functional molecule (e.g.,another peptide or protein). For example, a labeled antibody of theinvention can be derived by functionally linking an antibody of theinvention (by chemical coupling, genetic fusion, noncovalent associationor otherwise) to one or more other molecular entities, such as anotherantibody (e.g., a bispecific antibody or a diabody), a detectable agent,a pharmaceutical agent, and/or a protein or peptide that can mediateassociation of the antibody with another molecule (such as astreptavidin core region or a polyhistidine tag).

Useful detectable agents with which an antibody of the invention may bederivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. An antibody may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When an antibody is derivatized with a detectableenzyme, it is detected by adding additional reagents that the enzymeuses to produce a detectable reaction product. For example, when thedetectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. An antibody may also be derivatized withbiotin, and detected through indirect measurement of avidin orstreptavidin binding.

Another embodiment of the invention provides a crystallized antibody.According to one aspect, the invention relates to crystals of wholeanti-Aβ(20-42) globulomer antibodies and fragments thereof as disclosedherein, and formulations and compositions comprising such crystals.According to a further aspect, the crystallized antibody has a greaterhalf-life in vivo than the soluble counterpart of the antibody.According to a further aspect, the antibody retains biological activityafter crystallization.

Crystallized antibody of the invention may be produced according methodsknown in the art and as disclosed in WO02/072636, incorporated herein byreference.

Another embodiment of the invention provides a glycosylated antibodywherein the antibody comprises one or more carbohydrate residues.Nascent in vivo protein production may undergo further processing, knownas post-translational modification. In particular, sugar (glycosyl)residues may be added enzymatically, a process known as glycosylation.The resulting proteins bearing covalently linked oligosaccharide sidechains are known as glycosylated proteins or glycoproteins.

Antibodies are glycoproteins with one or more carbohydrate residues inthe Fc domain, as well as the variable domain. Carbohydrate residues inthe Fc domain have important effect on the effector function of the Fcdomain, with minimal effect on antigen binding or half-life of theantibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). Incontrast, glycosylation of the variable domain may have an effect on theantigen binding activity of the antibody. Glycosylation in the variabledomain may have a negative effect on antibody binding affinity, likelydue to steric hindrance (Co, M. S., et al., Mol. Immunol. (1993)30:1361-1367), or result in increased affinity for the antigen (Wallick,S. C., et al., Exp. Med. (1988) 168:1099-1109; Wright, A., et al., EMBOJ. (1991) 10:2717 2723).

One aspect of the present invention is directed to generatingglycosylation site mutants in which the O- or N-linked glycosylationsite of the antibody has been mutated. One skilled in the art cangenerate such mutants using standard well-known technologies. Thecreation of glycosylation site mutants that retain the biologicalactivity but have increased or decreased binding activity is anotherobject of the present invention.

In still another embodiment, the glycosylation of the antibody of theinvention is modified. For example, an aglycoslated antibody can be made(i.e., the antibody lacks glycosylation). Glycosylation can be alteredto, for example, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region glycosylation sites tothereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in International Appln. Publication No.WO03/016466A2, and U.S. Pat. Nos. 5,714,350 and 6,350,861, each of whichis incorporated herein by reference in its entirety.

Additionally or alternatively, a modified antibody of the invention canbe made that has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNAc structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the invention to therebyproduce an antibody with altered glycosylation. See, for example,Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana etal. (1999) Nat. Biotech. 17:176-1, as well as, European Patent NO.:EP1,176,195; International Appln. Publication Nos. WO03/035835 andWO99/54342 80, each of which is incorporated herein by reference in itsentirety.

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the invention may include, butare not limited to, glucose, galactose, mannose, fucose,n-acetylglucosamine and sialic acid. According to one aspect, theglycosylated antibody comprises glycosyl residues such that theglycosylation pattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies exhibiting human proteinglycosylation. For example, yeast strains have been genetically modifiedto express non-naturally occurring glycosylation enzymes such thatglycosylated proteins (glycoproteins) produced in these yeast strainsexhibit protein glycosylation identical to that of animal cells,especially human cells (U.S Patent Application Publication Nos.20040018590 and 20020137134; and WO05/100584).

Another embodiment is directed to an anti-idiotypic (anti-Id) antibodyspecific for such antibodies of the invention. An anti-Id antibody is anantibody, which recognizes unique determinants generally associated withthe antigen-binding region of another antibody. The anti-Id can beprepared by immunizing an animal with the antibody or a CDR containingregion thereof. The immunized animal will recognize, and respond to theidiotypic determinants of the immunizing antibody and produce an anti-Idantibody. The anti-Id antibody may also be used as an “immunogen” toinduce an immune response in yet another animal, producing a so-calledanti-anti-Id antibody.

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.According to a further aspect, the protein having a particularlyselected novel glycosylation pattern exhibits improved or alteredbiological properties.

D. Uses of Anti-Aβ(20-42) Globulomer Antibodies

Given their ability to bind to Aβ(20-42) globulomer, the anti-Aβ(20-42)globulomer antibodies, or antibodies against any other targeted Aβ form,of the invention can be used to detect Aβ(20-42) globulomer and/or anyother targeted Aβ form (e.g., in a biological sample such as serum, CSF,brain tissue or plasma), using a conventional immunoassay, such as anenzyme linked immunosorbent assays (ELISA), an radioimmunoassay (RIA) ortissue immunohistochemistry. The invention provides a method fordetecting Aβ(20-42) globulomer and/or any other targeted Aβ form in abiological sample comprising contacting a biological sample with anantibody of the invention and detecting either the antibody bound toAβ(20-42) globulomer (and/or any other targeted Aβ form) or unboundantibody, to thereby detect Aβ(20-42) globulomer, and/or any othertargeted Aβ form in the biological sample. The antibody is directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ³H, ¹⁴C,³⁵S, 90Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵ I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶ Ho, or ¹⁵³Sm.

Alternative to labeling the antibody, Aβ(20-42) globulomer and/or anyother targeted AB form can be assayed in biological fluids by acompetition immunoassay utilizing Aβ(20-42) globulomer standards labeledwith a detectable substance and an unlabeled anti-Aβ(20-42) globulomerantibody. In this assay, the biological sample, the labeled Aβ(20-42)globulomer standards and the anti-Aβ(20-42) globulomer antibody arecombined and the amount of labeled Aβ(20-42) globulomer standard boundto the unlabeled antibody is determined. The amount of Aβ(20-42)globulomer, and/or any other targeted Aβ form in the biological sampleis inversely proportional to the amount of labeled Aβ(20-42) globulomerstandard bound to the anti-Aβ(20-42) globulomer antibody.

According to one aspect of the invention, the antibodies of theinvention are capable of neutralizing Aβ(20-42) globulomer activity,and/or activity of any other targeted Aβ form both in vitro and in vivo.Accordingly, such antibodies of the invention can be used to inhibit(i.e. reduce) Aβ(20-42) globulomer activity, and/or activity of anyother targeted Aβ form, e.g., in a cell culture containing Aβ(20-42)globulomer, and/or any other targeted Aβ form in human subjects or inother mammalian subjects having Aβ(20-42) globulomer, and/or any othertargeted Aβ form with which an antibody of the invention cross-reacts.In one embodiment, the invention provides a method for inhibiting (i.e.reducing) Aβ(20-42) globulomer activity, and/or activity of any othertargeted Aβ form comprising contacting Aβ(20-42) globulomer, and/or anyother targeted Aβ form with an antibody of the invention such thatAβ(20-42) globulomer activity, and/or activity of any other targeted Aβform is inhibited (i.e. reduced). For example, in a cell culturecontaining, or suspected of containing Aβ(20-42) globulomer, and/or anyother targeted Aβ form an antibody of the invention can be added to theculture medium to inhibit (i.e. reduce) Aβ(20-42) globulomer activity,and/or activity of any other targeted Aβ form in the culture.

In another embodiment, the invention provides a method for inhibiting(i.e. reducing) activity of a targeted Aβ form in a subject,advantageously in a subject suffering from a disease or disorder inwhich activity of said Aβ form is detrimental, or a disease or disorderor disorder which is selected from the group consisting ofAlpha1-antitrypsin-deficiency, C1-inhibitor deficiency angioedema,Antithrombin deficiency thromboembolic disease, Kuru, Creutzfeld-Jacobdisease/scrapie, Bovine spongiform encephalopathy,Gerstmann-Straussler-Scheinker disease, Fatal familial insomnia,Huntington's disease, Spinocerebellar ataxia, Machado-Joseph atrophy,Dentato-rubro-pallidoluysian atrophy, Frontotemporal dementia, Sicklecell anemia, Unstable hemoglobin inclusion-body hemolysis, Drug-inducedinclusion body hemolysis, Parkinson's disease, Systemic AL amyloidosis,Nodular AL amyloidosis, Systemic AA amyloidosis, Prostatic amyloidosis,Hemodialysis amyloidosis, Hereditary (Icelandic) cerebral angiopathy,Huntington's disease, Familial visceral amyloidosis, Familial visceralpolyneuropathy, Familial visceral amyloidosis, Senile systemicamyloidosis, Familial amyloid neurophathy, Familial cardiac amyloidosis,Alzheimer's disease, Down syndrome, Medullary carcinoma thyroid and Type2 diabetes mellitus (T2DM).

The invention provides methods for inhibiting (i.e. reducing) theactivity of a targeted AB form in a subject suffering from such adisease or disorder, which method comprises administering to the subjectan antibody of the invention such that the activity of said Aβ form inthe subject is inhibited (i.e. reduced). In one aspect of the invention,said targeted Aβ form is a human Aβ form, and the subject is a humansubject. Alternatively, the subject can be a non-human mammal expressingAPP or any Aβ-form resulting in the generation of a targeted Aβ form towhich an antibody of the invention is capable of binding. Still furtherthe subject can be a non-human mammal into which a targeted Aβ form hasbeen introduced (e.g., by administration of the targeted Aβ form or byexpression of APP or any other Aβ-form resulting in the generation ofthe targeted Aβ form. An antibody of the invention can be administeredto a human subject for therapeutic purposes. Moreover, an antibody ofthe invention can be administered to a non-human mammal whereinexpression of APP or any Aβ-form resulting in the generation of atargeted Aβ form with which the antibody is capable of binding forveterinary purposes or as an animal model of human disease. Regardingthe latter, such animal models may be useful for evaluating thetherapeutic efficacy of antibodies of the invention (e.g., testing ofdosages and time courses of administration).

Another embodiment is a method for inhibiting (i.e. reducing) activityof a targeted Aβ form in a subject suffering from an amyloidosis, suchas Alzheimer's disease or Down syndrome.

A disorder in which activity of a targeted Aβ form is detrimentalincludes diseases and other disorders in which the presence of atargeted Aβ form in a subject suffering from the disorder has been shownto be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which activity ofa targeted Aβ form is detrimental is a disorder in which inhibition(i.e. reduction) of the activity said Aβ form is expected to alleviatesome or all of the symptoms and/or progression of the disorder. Suchdisorders may be evidenced, for example, by an increase in theconcentration of a targeted Aβ form in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofthe targeted AB form in serum, brain tissue, plasma, cerebrospinalfluid, etc. of the subject), which can be detected, for example, usingan anti-Aβ(20-42) globulomer antibody and/or antibody against any othertargeted Aβ form as described above or any antibody to any Aβ form thatcomprises the globulomer epitope with which the antibodies of thepresent invention are reactive. Non-limiting examples of disorders thatcan be treated with the antibodies of the invention include thosedisorders disclosed herein and those discussed in the section belowpertaining to pharmaceutical compositions of the antibodies of theinvention.

In still yet another embodiment, the present invention relates to amethod for preventing the progression (e.g., worsening) of a diseasecondition described herein. The method comprises administering to thesubject in need of treatment thereof (e.g., a mammal, such as a human) atherapeutically effective amount of any of the binding proteins orantibodies as described herein. Alternatively, the method comprisesadministering to the subject a therapeutically effective amount of anyof the proteins as described herein, in combination with atherapeutically effective amount of at least one therapeutic agent.

In the above described methods for preventing the development orprogression of a disorder described herein one or more biomarkers,diagnostic tests or combination of biomarkers and diagnostic tests knownto those skilled the art can be used to determine whether or not (1) asubject is at risk of developing one or more of the disorders describedherein; or (2) the disorders described herein in the subject previouslydiagnosed with one or more of the aforementioned disorders isprogressing (e.g., worsening).

One or more biomarkers, diagnostic tests or combinations of biomarkersand diagnostic tests known in the art can be used to identify subjectswho are at risk of developing a disorder described herein. Likewise, oneor more biomarkers, diagnostic tests or combinations of biomarkers anddiagnostic tests known in the art can be used to determine theprogression of the disease or condition of subjects who have beenidentified as suffering from a disorder described herein. For example,one or more biological markers, neuroimaging markers or combination ofbiological or neuroimaging markers (e.g., MRI, etc.) can be used toidentify subjects at risk of developing Alzheimer's disease or, forthose subjects identified as suffering from Alzheimer's disease, theprogression of the disease. Biological markers that can be examinedinclude, but are not limited to, beta-amyloid₁₋₄₂, tau, phosphorylatedtau (ptau), plasma Aβ antibodies, α-antichymotrypsin, amyloid precursorprotein, APP isoform ratio in platelets, β-secretase (also known asBACE), CD59, 8-hydroxy-deoxyguanine, glutamine synthetase, glialfibrillary acidic protein (GFAP), antibodies to GFAP,interleukin-6-receptor complex, kallikrein, melanotransferrin,neurofilament proteins, nitrotyrosine, oxysterols, sulphatides, synapticmarkers, S100β, NPS, plasma signaling proteins, etc., or anycombinations thereof (See, Shaw, L., et al., Nature Reviews 2007, 6,295-303. Borroni, B., et al., Current Med. Chem. 2007, 14, 1171-1178.Phillips, K., et al., Nature Reviews 2006, 5 463-469. Bouwman, F. H., etal., Neurology 2007, 69, 1006-1011; Ray, S., et al., Nature Medicine2007, 13(11), 1359-1362. Cummings, J., et al., Neurology 2007, 69,1622-1634.).

E. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising anantibody of the invention and a pharmaceutically acceptable carrier. Thepharmaceutical compositions comprising antibodies of the invention arefor use in, but not limited to, diagnosing, detecting, or monitoring adisorder, in preventing, treating, managing, or ameliorating of adisorder or one or more symptoms thereof, and/or in research. In aspecific embodiment, a composition comprises one or more antibodies ofthe invention. In another embodiment, the pharmaceutical compositioncomprises one or more antibodies of the invention and one or moreprophylactic or therapeutic agents other than antibodies of theinvention for treating a disorder in which activity of a targeted Aβform is detrimental. In a further embodiment, the prophylactic ortherapeutic agents are known to be useful for, or have been, or arecurrently being used in the prevention, treatment, management, oramelioration of a disorder, or one or more symptoms thereof. Inaccordance with these embodiments, the composition may further compriseof a carrier, diluent or excipient.

The antibodies of the invention can be incorporated into pharmaceuticalcompositions suitable for administration to a subject. Typically, thepharmaceutical composition comprises an antibody of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody.

In a further embodiment, the pharmaceutical composition comprises atleast one additional therapeutic agent for treating a disorder asdisclosed herein.

Various delivery systems are known and can be used to administer one ormore antibodies of the invention or the combination of one or moreantibodies of the invention and a prophylactic agent or therapeuticagent useful for preventing, managing, treating, or ameliorating adisorder or one or more symptoms thereof, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the antibody or antibody fragment, receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a prophylactic or therapeutic agent of theinvention include, but are not limited to, parenteral administration(e.g., intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidurala administration, intratumoral administration,and mucosal administration (e.g., intranasal and oral routes). Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. See,e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO97/32572, WO97/44013, WO98/31346, and WO99/66903, each ofwhich is incorporated herein by reference their entireties. In oneembodiment, an antibody of the invention, combination therapy, or acomposition of the invention is administered using Alkermes AIR®pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.).In a specific embodiment, prophylactic or therapeutic agents of theinvention are administered intramuscularly, intravenously,intratumorally, orally, intranasally, pulmonary, or subcutaneously. Theprophylactic or therapeutic agents may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theantibodies of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion, by injection, or by means of an implant, said implantbeing of a porous or non-porous material, including membranes andmatrices, such as sialastic membranes, polymers, fibrous matrices (e.g.,Tissuel®), or collagen matrices. In one embodiment, an effective amountof one or more antibodies of the invention is administered locally tothe affected area to a subject to prevent, treat, manage, and/orameliorate a disorder or a symptom thereof. In another embodiment, aneffective amount of one or more antibodies of the invention isadministered locally to the affected area in combination with aneffective amount of one or more therapies (e.g., one or moreprophylactic or therapeutic agents) other than an antibody of theinvention of a subject to prevent, treat, manage, and/or ameliorate adisorder or one or more symptoms thereof.

In another embodiment, the antibody can be delivered in a controlledrelease or sustained release system. In one embodiment, a pump may beused to achieve controlled or sustained release (see Langer, supra;Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980,Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). Inanother embodiment, polymeric materials can be used to achievecontrolled or sustained release of the therapies of the invention (seee.g., Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 7 1:105); U.S. Pat. No. 5,679,377; U.S. Pat. No.5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat.No. 5,128,326; PCT Publication No. WO99/15154; and PCT Publication No.WO99/20253. Examples of polymers used in sustained release formulationsinclude, but are not limited to, poly(2-hydroxy ethyl methacrylate),poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinylacetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. In a particular embodiment, the polymerused in a sustained release formulation is inert, free of leachableimpurities, stable on storage, sterile, and biodegradable. In yetanother embodiment, a controlled or sustained release system can beplaced in proximity of the prophylactic or therapeutic target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore antibodies of the invention. See, e.g., U.S. Pat. No. 4,526,938,PCT publication WO91/05548, PCT publication WO96/20698, Ning et al.,1996, “Intratumoral Radioimmunotheraphy of a Human Colon CancerXenograft Using a Sustained-Release Gel,” Radiotherapy &Oncology39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting ofLong-Circulating Emulsions,” PDA Journal of Pharmaceutical Science &Technology 50:372-397, Cleek et al., 1997, “Biodegradable PolymericCarriers for a bFGF Antibody for Cardiovascular Application,” Pro.Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al.,1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibodyfor Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, each of which is incorporated herein by reference in theirentireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see, e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868).Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral, e.g.,intravenous, intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If the compositions of the invention are to be administered topically,the compositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19^(th) ed., Mack Pub. Co., Easton, Pa.(1995). For non-sprayable topical dosage forms, viscous to semi-solid orsolid forms comprising a carrier or one or more excipients compatiblewith topical application and having a dynamic viscosity greater thanwater are typically employed. Suitable formulations include, withoutlimitation, solutions, suspensions, emulsions, creams, ointments,powders, liniments, salves, and the like, which are, if desired,sterilized or mixed with auxiliary agents (e.g., preservatives,stabilizers, wetting agents, buffers, or salts) for influencing variousproperties, such as, for example, osmotic pressure. Other suitabletopical dosage forms include sprayable aerosol preparations wherein theactive ingredient, for example in combination with a solid or liquidinert carrier, is packaged in a mixture with a pressurized volatile(e.g., a gaseous propellant, such as freon) or in a squeeze bottle.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well-known in the art.

If the method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If the method of the invention comprises oral administration,compositions can be formulated orally in the form of tablets, capsules,cachets, gelcaps, solutions, suspensions, and the like. Tablets orcapsules can be prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers(e.g., lactose, microcrystalline cellulose, or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, or silica);disintegrants (e.g., potato starch or sodium starch glycolate); orwetting agents (e.g., sodium lauryl sulphate). The tablets may be coatedby methods well-known in the art. Liquid preparations for oraladministration may take the form of, but not limited to, solutions,syrups or suspensions, or they may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

The method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, an antibody of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR® pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass.).

The method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use. The methods ofthe invention may additionally comprise of administration ofcompositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

The methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, compositioncan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theantibodies, or pharmaceutical compositions, of the invention is packagedin a hermetically sealed container such as an ampoule or sachetteindicating the quantity of the antibody. In one embodiment, one or moreof the antibodies, or pharmaceutical compositions of the invention issupplied as a dry sterilized lyophilized powder or water freeconcentrate in a hermetically sealed container and can be reconstituted(e.g., with water or saline) to the appropriate concentration foradministration to a subject. In one embodiment, one or more of theantibodies or pharmaceutical compositions of the invention is suppliedas a dry sterile lyophilized powder in a hermetically sealed containerat a unit dosage of at least 5 mg, for example at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized antibodies orpharmaceutical compositions of the invention should be stored at between2° C. and 8° C. in its original container and the antibodies, orpharmaceutical compositions of the invention should be administeredwithin 1 week, for example within 5 days, within 72 hours, within 48hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours,within 3 hours, or within 1 hour after being reconstituted. In analternative embodiment, one or more of the antibodies or pharmaceuticalcompositions of the invention is supplied in liquid form in ahermetically sealed container indicating the quantity and concentrationof the antibody. In a further embodiment, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, for example at least 0.5 mg/ml, at least 1 mg/ml,at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least75 mg/ml or at least 100 mg/ml. The liquid form should be stored atbetween 2° C. and 8° C. in its original container.

The antibodies of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration. Inone aspect, antibodies will be prepared as an injectable solutioncontaining 0.1-250 mg/ml antibody. The injectable solution can becomposed of either a liquid or lyophilized dosage form in a flint oramber vial, ampule or pre-filled syringe. The buffer can be L-histidine(1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Othersuitable buffers include but are not limited to, sodium succinate,sodium citrate, sodium phosphate or potassium phosphate. Sodium chloridecan be used to modify the toxicity of the solution at a concentration of0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectantscan be included for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trehaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-Methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRIJ surfactants. The pharmaceutical compositioncomprising the antibodies of the invention prepared as an injectablesolution for parenteral administration, can further comprise an agentuseful as an adjuvant, such as those used to increase the absorption, ordispersion of the antibody. A particularly useful adjuvant ishyaluronidase, such as Hylenex® (recombinant human hyaluronidase).Addition of hyaluronidase in the injectable solution improves humanbioavailability following parenteral administration, particularlysubcutaneous administration. It also allows for greater injection sitevolumes (i.e. greater than 1 ml) with less pain and discomfort, andminimum incidence of injection site reactions. (See International Appln.Publication No. WO 04/078140 and U.S. Patent Appln. Publication No.US2006104968, incorporated herein by reference.)

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Compositions can be in the form of injectableor infusible solutions, such as compositions similar to those used forpassive immunization of humans with other antibodies. In one embodiment,the antibody is administered by intravenous infusion or injection. Inanother embodiment, the antibody is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e., abinding protein, e.g. an antibody, of the present invention) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile, lyophilized powders for the preparation ofsterile injectable solutions, methods of preparation comprise vacuumdrying and spray-drying that yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including, in the composition, anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art. For many therapeutic applications, theroute/mode of administration may be subcutaneous injection, intravenousinjection or infusion. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The antibody (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the antibody may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer an antibody of the invention by other than parenteraladministration, it may be necessary to coat the antibody with, orco-administer the antibody with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody of the invention iscoformulated with and/or coadministered with one or more additionaltherapeutic agents that are useful for treating disorders or diseasesdescribed herein. For example, an anti-Aβ(20-42) globulomer antibody ofthe invention may be coformulated and/or coadministered with one or moreadditional antibodies that bind other targets (e.g., antibodies thatbind other soluble antigens or that bind cell surface molecules).Furthermore, one or more antibodies of the invention may be used incombination with two or more of the foregoing therapeutic agents. Suchcombination therapies may advantageously utilize lower dosages of theadministered therapeutic agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies.

In certain embodiments, an antibody of the invention is linked to ahalf-life extending vehicle known in the art. Such vehicles include, butare not limited to, the Fc domain, polyethylene glycol, and dextran.Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082 and published PCT Application No. WO 99/25044, which arehereby incorporated by reference for any purpose.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding an antibody of the invention are administered totreat, prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded antibody of the invention that mediates aprophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, Science 260:926-932(1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217;May, 1993, TIBTECH 11(5):155-215. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); and Kriegler, Gene Transfer and Expression, A LaboratoryManual, Stockton Press, NY (1990). Detailed description of variousmethods of gene therapy are disclosed in US20050042664 A1 which isincorporated herein by reference.

Antibodies of the invention can be used alone or in combination to treatdiseases such as Alzheimer's disease, Down syndrome, dementia,Parkinson's disease, or any other disease or condition associated with abuild up of amyloid beta protein within the brain. The antibodies of thepresent invention may be used to treat “conformational diseases”. Suchdiseases arise from secondary to tertiary structural changes withinconstituent proteins with subsequent aggregation of the altered proteins(Hayden et al., JOP. J Pancreas 2005; 6(4):287-302). In particular, theantibodies of the present invention may be used to treat one or more ofthe following conformational diseases: Alpha1-antitrypsin-deficiency,C1-inhibitor deficiency angioedema, Antithrombin deficiencythromboembolic disease, Kuru, Creutzfeld-Jacob disease/scrapie, Bovinespongiform encephalopathy, Gerstmann-Straussler-Scheinker disease, Fatalfamilial insomnia, Huntington's disease, Spinocerebellar ataxia,Machado-Joseph atrophy, Dentato-rubro-pallidoluysian atrophy,Frontotemporal dementia, Sickle cell anemia, Unstable hemoglobininclusion-body hemolysis, Drug-induced inclusion body hemolysis,Parkinson's disease, Systemic AL amyloidosis, Nodular AL amyloidosis,Systemic AA amyloidosis, Prostatic amyloidosis, Hemodialysisamyloidosis, Hereditary (Icelandic) cerebral angiopathy, Huntington'sdisease, Familial visceral amyloidosis, Familial visceralpolyneuropathy, Familial visceral amyloidosis, Senile systemicamyloidosis, Familial amyloid neurophathy, Familial cardiac amyloidosis,Alzheimer's disease, Down syndrome, Medullary carcinoma thyroid and Type2 diabetes mellitus (T2DM) Preferably, the antibodies of the presentinvention may be utilized to treat an amyloidosis, for example,Alzheimer's disease and Down syndrome.

It should be understood that the antibodies of the invention can be usedalone or in combination with one or more additional agents, e.g., atherapeutic agent (for example, a small molecule or biologic), saidadditional agent being selected by the skilled artisan for its intendedpurpose. For example, the additional therapeutic agent can be a“cognitive enhancing drug,” which is a drug that improves impaired humancognitive abilities of the brain (namely, thinking, learning, andmemory). Cognitive enhancing drugs work by altering the availability ofneurochemicals (e.g., neurotransmitters, enzymes, and hormones), byimproving oxygen supply, by stimulating nerve growth, or by inhibitingnerve damage. Examples of cognitive enhancing drugs include a compoundthat increases the activity of acetylcholine such as, but not limitedto, an acetylcholine receptor agonist (e.g., a nicotinic α-7 receptoragonist or allosteric modulator, an α4β2 nicotinic receptor agonist orallosteric modulators), an acetylcholinesterase inhibitor (e.g.,donepezil, rivastigmine, and galantamine), a butyrylcholinesteraseinhibitor, an N-methyl-D-aspartate (NMDA) receptor antagonist (e.g.,memantine), an activity-dependent neuroprotective protein (ADNP)agonist, a serotonin 5-HT1A receptor agonist (e.g., xaliproden), a 5-HT₄receptor agonist, a 5-HT₆ receptor antagonist, a serotonin 1A receptorantagonist, a histamine H₃ receptor antagonist, a calpain inhibitor, avascular endothelial growth factor (VEGF) protein or agonist, a trophicgrowth factor, an anti-apoptotic compound, an AMPA-type glutamatereceptor activator, a L-type or N-type calcium channel blocker ormodulator, a potassium channel blocker, a hypoxia inducible factor (HIF)activator, a HIF prolyl 4-hydroxylase inhibitor, an anti-inflammatoryagent, an inhibitor of amyloid Aβ peptide or amyloid plaque, aninhibitor of tau hyperphosphorylation, a phosphodiesterase 5 inhibitor(e.g., tadalafil, sildenafil), a phosphodiesterase 4 inhibitor, amonoamine oxidase inhibitor, or pharmaceutically acceptable saltthereof. Specific examples of such cognitive enhancing drugs include,but are not limited to, cholinesterase inhibitors such as donepezil(Aricept®), rivastigmine (Exelon®), galanthamine (Reminyl®),N-methyl-D-aspartate antagonists such as memantine (Namenda®). At leastone cognitive enhancing drug can be administered simultaneously with theantibodies of the present invention or sequentially with the antibodiesof the present invention (and in any order) including those agentscurrently recognized, or in the future being recognized, as useful totreat the disease or condition being treated by an antibody of thepresent invention). Additionally, it is believed that the combinationsdescribed herein may have additive or synergistic effects when used inthe above-described treatment. The additional agent also can be an agentthat imparts a beneficial attribute to the therapeutic composition,e.g., an agent that affects the viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth above are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can comprise an antibody of the presentinvention and at least one additional agent selected from the listsbelow. The combination can also include more than one additional agent,e.g., two or three additional agents if the combination is such that theformed composition can perform its intended function.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody of the invention. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the antibody may be determined by aperson skilled in the art and may vary according to factors such as thedisease state, age, sex, and weight of the individual, and the abilityof the antibody to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody of the invention is0.1-20 mg/kg, for example 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1: Preparation of Globulomers

a) Aβ(1-42) Globulomer:

The Aβ(1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland)was suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6mg/ml and incubated for complete solubilization under shaking at 37° C.for 1.5 h. The HFIP acts as a hydrogen-bond breaker and is used toeliminate pre-existing structural inhomogeneities in the Aβ peptide.HFIP was removed by evaporation in a SpeedVac and Aβ(1-42) resuspendedat a concentration of 5 mM in dimethylsulfoxide and sonicated for 20 s.The HFIP-pre-treated Aβ(1-42) was diluted in phosphate-buffered saline(PBS) (20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume 2%sodium dodecyl sulfate (SDS) (in H₂O) added (final concentration of 0.2%SDS). An incubation for 6 h at 37° C. resulted in the 16/20-kDa Aβ(1-42)globulomer (short form for globular oligomer) intermediate. The38/48-kDa Aβ(1-42) globulomer was generated by a further dilution withthree volumes of H₂O and incubation for 18 h at 37° C. Aftercentrifugation at 3000 g for 20 min the sample was concentrated byultrafiltration (30-kDa cut-off), dialysed against 5 mM NaH₂PO₄, 35 mMNaCl, pH 7.4, centrifuged at 10,000 g for 10 min and the supernatantcomprising the 38/48-kDa Aβ(1-42) globulomer withdrawn. As analternative to dialysis the 38/48-kDa Aβ(1-42) globulomer could also beprecipitated by a ninefold excess (v/v) of ice-cold methanol/acetic acidsolution (33% methanol, 4% acetic acid) for 1 h at 4° C. The 38/48-kDaAβ(1-42) globulomer is then pelleted (10 min at 16200 g), resuspended in5 mM NaH₂PO₄, 35 mM NaCl, pH 7.4, and the pH adjusted to 7.4.

b) Aβ(20-42) Globulomer:

1.59 ml of Aβ(1-42) globulomer preparation prepared according to Example1a were admixed with 38 ml of buffer (50 mM MES/NaOH, pH 7.4) and 200 μlof a 1 mg/ml thermolysin solution (Roche) in water. The reaction mixturewas stirred at RT for 20 h. Then, 80 μl of a 100 mM EDTA solution, pH7.4, in water were added and the mixture was furthermore adjusted to anSDS content of 0.01% with 400 μl of a 1% strength SDS solution. Thereaction mixture was concentrated to approximately 1 ml via a 15 ml 30kDa Centriprep tube. The concentrate was admixed with 9 ml of buffer (50mM MES/NaOH, 0.02% SDS, pH 7.4) and again concentrated to 1 ml. Theconcentrate was dialyzed at 6° C. against 1 l of buffer (5 mM sodiumphosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate wasadjusted to an SDS content of 0.1% with a 2% strength SDS solution inwater. The sample was centrifuged at 10,000 g for 10 min and theAβ(20-42) globulomer supernatant was withdrawn.

c) Aβ(12-42) Globulomer:

2 ml of an Aβ(1-42) globulomer preparation prepared according to Example1a were admixed with 38 ml buffer (5 mM sodium phosphate, 35 mM sodiumchloride, pH 7.4) and 150 μl of a 1 mg/ml GluC endoproteinase (Roche) inwater. The reaction mixture was stirred for 6 h at RT, and a further 150μl of a 1 mg/ml GluC endoproteinase (Roche) in water were subsequentlyadded. The reaction mixture was stirred at RT for another 16 h, followedby addition of 8 μl of a 5 M DIFP solution. The reaction mixture wasconcentrated to approximately 1 ml via a 15 ml 30 kDa Centriprep tube.The concentrate was admixed with 9 ml of buffer (5 mM sodium phosphate,35 mM sodium chloride, pH 7.4) and again concentrated to 1 ml. Theconcentrate was dialyzed at 6° C. against 1 l of buffer (5 mM sodiumphosphate, 35 mM NaCl) in a dialysis tube for 16 h. The dialysate wasadjusted to an SDS content of 0.1% with a 1% strength SDS solution inwater. The sample was centrifuged at 10,000 g for 10 min and theAβ(12-42) globulomer supernatant was withdrawn.

d) Cross-Linked Aβ(1-42) Globulomer:

The Aβ(1-42) synthetic peptide (H-1368, Bachem, Bubendorf, Switzerland)was suspended in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6mg/ml and incubated for complete solubilization under shaking at 37° C.for 1.5 h. The HFIP acts as a hydrogen-bond breaker and was used toeliminate pre-existing structural inhomogeneities in the Aβ peptide.HFIP was removed by evaporation by a SpeedVac and Aβ(12-42) globulomerAβ(1-42) resuspended at a concentration of 5 mM in dimethylsulfoxide andsonicated for 20 s. The HFIP-pre-treated Aβ(1-42) was diluted in PBS (20mM NaH₂PO₄, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 vol. 2% SDS (inwater) added (final conc. Of 0.2% SDS). An incubation for 6 h at 37° C.resulted in the 16/20-kDa Aβ(1-42) globulomer (short form for globulomeroligomer) intermediate. The 38/48-kDa Aβ(1-42) globulomer was generatedby a further dilution with 3 volumes of water and incubation for 18 h at37° C. Cross-linking of the 38/48-kDa Aβ(1-42) globulomer was nowperformed by incubation with 1 mM glutaraldehyde for 2 h at 21° C. roomtemperature followed by ethanolamine (5 mM) treatment for 30 min at roomtemperature.

Example 2: Generation, Isolation and Characterization of HumanizedAnti-Aβ(20-42) Globulomer Antibodies Example 2.1: Selection of HumanAntibody Frameworks

Selection of human antibody frameworks was based on similarity ofcanonical structures and amino acid sequence homology of humanantibodies. Further, the retention of amino acid residues which supportloop structures and VH/VL interface as well as the retention of aminoacid residues of the Vernier zone was taken into account whenidentifying suitable acceptor VL and VH framework sequences based onamino acid sequence homology of human VH and Vκ germline sequences.Moreover, immunogenicity of VH and VL sequences resulting from grafting4D10 CDRs into potentially suitable acceptor VL and VH frameworksequences was evaluated in silico based on the predicted affinity ofoverlapping peptides to a variety of MHC class I and/or MHC class IIalleles. VH and VL were adapted to the consensus of the respective VH orVL family to further minimize potential immunogenicity. Selectedbackmutations to murine amino acid residues were performed to retainamino acids which support loop structures and VH/VL interface. Thefrequencies of these backmutations in corresponding pools of naturallyoccurring human VH or VL sequences having the respective VH or VLgermline gene were determined by amino acid sequence alignments. The VHand VL sequences resulting from the considerations described above werechecked for potential N-linked glycosylation sites (NXS or NXT, whereinX is any amino acid except P).

Example 2.2: Humanization of Murine Anti-Aβ(20-42) Globulomer Antibody

4D10hum_VH.1z (SEQ ID NO:4): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH3-53 and JH6 sequences.

4D10hum_VH.1 (SEQ ID NO:5): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH3-53 and JH6 sequencescomprising VH3 consensus change I12V.

4D10hum_VH.1a (SEQ ID NO:6): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH3-53 and JH6 sequencescomprising VH3 consensus change I12V and framework backmutations A24V,V29L, V48L, S49G, F67L, R71K, N76S and L78V.

4D10hum_VH.1b (SEQ ID NO:7): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH3-53 and JH6 sequencescomprising backmutations V29L and R71K. 4D10hum_VH.2z (SEQ ID NO:8): Theheavy chain CDR sequences from the murine anti-Aβ(20-42) globulomerantibody 4D10 described in Table 4 were grafted into an acceptorframework of human VH4-59 and JH6 sequences.

4D10hum_VH.2 (SEQ ID NO:9): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH4-59 and JH6 sequencescomprising a Q1E change to prevent N-terminal pyroglutamate formation.

4D10hum_VH.2a (SEQ ID NO:10): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH4-59 and JH6 sequencescomprising a Q1E change to prevent N-terminal pyroglutamate formation,and framework backmutations G27F, I29L, I37V, I48L, V67L, V71K, N76S andF78V.

4D10hum_VH.2b (SEQ ID NO:11): The heavy chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human VH4-59 and JH6 sequencescomprising a Q1E change to prevent N-terminal pyroglutamate formation,and framework backmutations G27F, I29L and V71K.

4D10hum_Vκ.1z (SEQ ID NO:12): The light chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human Vκ A17/2-30 and Jκ2sequences.

4D10hum_Vκ.1 (SEQ ID NO:13): The light chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human Vκ A17/2-30 and Jκ2sequences comprising Vκ2 consensus changes S7T, L15P, Q37L, R39K andR45Q.

4D10hum_Vκ.1a (SEQ ID NO:14): The light chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human Vκ A17/2-30 and Jκ2sequences comprising Vκ2 consensus changes S7T, L15P, Q37L, R39K andR45Q, and framework backmutation F36L which affects the VL/VH interface.

4D10hum_Vκ.1b (SEQ ID NO:15): The light chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human Vκ A17/2-30 and Jκ2sequences comprising Vκ2 consensus changes S7T and Q37L.

4D10hum_Vκ.1c (SEQ ID NO:16): The light chain CDR sequences from themurine anti-Aβ(20-42) globulomer antibody 4D10 described in Table 4 weregrafted into an acceptor framework of human Vκ A17/2-30 and Jκ2sequences comprising Vκ2 consensus changes S7T, Q37L and R39K.

Some of said VH and Vκ back-mutations, consensus changes or the Q1Emutation in 4D10hum_VH.2, 4D10hum_VH.2a or 4D10hum_VH.2b may be removedduring a subsequent affinity maturation.

Example 2.3: Construction of Humanized Antibodies

In silico constructed humanized antibodies described above will beconstructed de novo using oligonucleotides. For each variable regioncDNA, 6 oligonucleotides of 60-80 nucleotides each will be designed tooverlap each other by 20 nucleotides at the 5′ and/or 3′ end of eacholigonucleotide. In an annealing reaction, all 6 oligos will becombined, boiled, and annealed in the presence of dNTPs. Then DNApolymerase I, Large (Klenow) fragment (New England Biolabs #M0210,Beverley, Mass.) will be added to fill-in the approximately 40 bp gapsbetween the overlapping oligonucleotides. PCR will then be performed toamplify the entire variable region gene using two outermost primerscontaining overhanging sequences complementary to the multiple cloningsite in a modified pBOS vector (Mizushima, S. and Nagata, S., (1990)Nucleic acids Research Vol 18, No. 17)). The PCR products derived fromeach cDNA assembly will be separated on an agarose gel and the bandcorresponding to the predicted variable region cDNA size will be excisedand purified. The variable heavy region will be inserted in-frame onto acDNA fragment encoding the human IgG1 constant region containing 2hinge-region amino acid mutations by homologous recombination inbacteria. These mutations are a leucine to alanine change at position234 (EU numbering) and a leucine to alanine change at position 235 (Lundet al., 1991, J. Immunol., 147:2657). The variable light chain regionwill be inserted in-frame with the human kappa constant region byhomologous recombination. Bacterial colonies will be isolated andplasmid DNA extracted; cDNA inserts will be sequenced in their entirety.Correct humanized heavy and light chains corresponding to each antibodywill be co-transfected into COS cells to transiently produce full-lengthhumanized anti-Aβ globulomer antibodies. Cell supernatants containingrecombinant chimeric antibody will be purified by Protein A Sepharosechromatography and bound antibody will be eluted by addition of acidbuffer. Antibodies will be neutralized and dialyzed into PBS. (DiederMoechars et al J Biol Chem 274:6483-6492 (1999); Ausubel, F. M. et al.eds., Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley &Sons, NY. (ISBN 0-471-32938-X); Lu and Weiner eds., Cloning andExpression Vectors for Gene Function Analysis (2001) BioTechniquesPress. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X); Kontermann andDubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790pp. (ISBN 3-540-41354-5); Old, R. W. & S. B. Primrose, Principles ofGene Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985)Blackwell Scientific Publications, Boston. Studies in Microbiology;V.2:409 pp. (ISBN 0-632-01318-4); Sambrook, J. et al. eds., MolecularCloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor LaboratoryPress, NY. Vols. 1-3. (ISBN 0-87969-309-6); Winnacker, E. L. From GenesTo Clones: Introduction To Gene Technology (1987) VCH Publishers, NY(translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4); all ofwhich are incorporated by reference in their entirety).

Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the present disclosure or the invention as defined in theappended claims.

Example 2:4: Expression and Purification of Humanized Antibodies inHEK293 Cells

DNA constructs encoding an antibody heavy chain as set forth in SEQ IDNO:46; an antibody heavy chain as set forth in SET ID NO:47, and anantibody light chain construct encoding a polypeptide as set forth inSEQ ID NO:48 were prepared as described in example 2.3. After DNAconfirmation by sequencing, all heavy chain and light chain DNAconstructs were expanded in E. coli and DNA was purified using QiagenEndo Free Plasmid Maxi Prep (cat#12362, QIAGEN) according to themanufacturer's protocol.

For expression of a monoclonal antibody 4D10hum#1, HEK293 (EBNA) cellswere transiently cotransfected with plasmids encoding the heavy chainset forth in SEQ ID NO:46 and the light chain set forth in SEQ ID NO:48.For expression of a monoclonal antibody 4D10hum#2, HEK293 (EBNA) cellswere transiently cotransfected with plasmids encoding the heavy chainset forth in SEQ ID NO:47 and the light chain set forth in SEQ ID NO:48.Before transfection, HEK293 (EBNA) cells were propagated in Freestyle293 media (Invitrogen, Carlsbad Calif.) at a 0.5 l scale in cultureflasks (2 L Corning Cat#431198) shaking in a CO₂ incubator (8% CO₂, 125rpm, 37° C.). When the cell cultures reached a density of 1×10⁶cells/ml, cells were transfected by adding transfection complex. Thetransfection complex was prepared by first mixing 150 μg of the plasmidencoding the light chain, 100 μg of the plasmid encoding the heavy chainand 25 ml Freestyle medium, followed by the addition of 500 μl PEIsolution (1 mg/ml (pH 7.0) linear 25 kDa polyethylenimine, PolysciencesCat#23966). The transfection complex was mixed by inversion andincubated at room temperature for 15 min prior to being added to thecell culture. After transfection, cultures continued to be grown in theCO₂ incubator (8% CO₂, 125 rpm, 37° C.). Twenty-four hours aftertransfection, the culture media were supplemented with 50 ml of a 5%Tryptone N1 solution (Organo Technie, La Courneuve France Cat#19553).Six days after transfection, the cells were pelleted by centrifugation(16,000 g, 30 min), the supernatant containing the expressed antibodieswas sterile filtered (0.2 μm PES filter) and placed at 4° C. untilinitiation of the purification step. The expressed antibodies werepurified from the supernatants by Protein A sepharose affinitychromatography using Pierce Thermo Scientific reagents and protocolaccording to manufacturer's instructions. The protein eluates weredialyzed against PBS (pH 7). The purified 4D10hum antibodies werespectrophotometrically quantified at 280 nm, and analyzed by massspectrometry and size exclusion chromatography (SEC).

Example 2:5: Affinity Analysis of Humanized Antibodies

Interaction of the purified humanized antibodies 4D10hum#1 and 4D10hum#2to Aβ(20-42) globulomer was evaluated by surface plasmon resonance (SPR)analysis using a BIAcore device. Goat anti-human IgG Fc (10,000 RU) wasdirectly immobilized on a CMS sensor chip by an amine coupling procedureaccording to the manufacturer's instructions (BIAcore). The respective4D10hum antibody was captured on the goat anti-human IgG Fc coatedsurface of the chip by injecting 5.0 μl of a 1 μg/ml 4D10hum antibodysolution at a flow rate of 10-15 μl/min. Interaction of solubleAβ(20-42) globulomer with the 4D10hum antibody on the sensor chip wasexamined by injecting globulomer solutions (concentration range:20-0.3125 nM) at a flow rate of 50 μl/min. The association rate wasmonitored for 5.0 min and the dissociation rate was monitored for 10min. From the resulting sensorgrams the association rate constant(k_(on)), dissociation rate constant (k_(off)) and equilibriumdissociation constant (K_(D)) were determined using to themanufacturer's software and instructions. The kinetic and equilibriumconstants determined for three different preparations of 4D10hum#1 andtwo different preparations of 4D10hum#2 are summarized in table 7. Table7 also shows affinity data of antibodies #3, #4 and #5 having chimericand humanized chains. The heavy chains of antibodies #4 and #5 are as in4D10hum#1 or #2, and the light chains are chimeras of m4D10 VL (SEQ IDNO:24) and the human Ig kappa constant region (SEQ ID NO:27). The lightchains of antibody #3 are as in 4D10hum#1 and #2, and the heavy chainsare chimeras of m4D10 VH (SEQ ID NO23) and the human Ig gamma-1 constantregion (SEQ ID NO:25).

TABLE 7 AFFINITY OF 4D10HUM ANTIBODIES FOR Aβ(20-42) GLOBULOMER k_(on)k_(off) K_(D) Antibody Antibody Lot Experiment [M⁻¹s⁻¹] [s⁻¹] [M]4D10hum#1 #1759115 1 5.22 × 10⁵ 3.02 × 10⁻⁴ 5.78 × 10⁻¹⁰ 2 5.39 × 10⁵3.61 × 10⁻⁴ 6.71 × 10⁻¹⁰ average 5.31 × 10⁵ 3.32 × 10⁻⁴ 6.25 × 10⁻¹⁰#1763976 1 4.86 × 10⁵ 2.81 × 10⁻⁴ 5.78 × 10⁻¹⁰ 2 5.13 × 10⁵ 3.04 × 10⁻⁴5.93 × 10⁻¹⁰ average 5.00 × 10⁵ 2.93 × 10⁻⁴ 5.86 × 10⁻¹⁰ #1773662 1 4.66× 10⁵ 2.49 × 10⁻⁴ 5.35 × 10⁻¹⁰ 2 5.18 × 10⁵ 2.87 × 10⁻⁴ 5.53 × 10⁻¹⁰average 4.92 × 10⁵ 2.68 × 10⁻⁴ 5.44 × 10⁻¹⁰ 4D10hum#2 #1759119 1 5.93 ×10⁵ 2.70 × 10⁻⁴ 4.54 × 10⁻¹⁰ 2 5.46 × 10⁵ 3.32 × 10⁻⁴ 6.09 × 10⁻¹⁰average 5.70 × 10⁵ 3.01 × 10⁻⁴ 5.32 × 10⁻¹⁰ #1773659 1 5.07 × 10⁵ 2.68 ×10⁻⁴ 5.29 × 10⁻¹⁰ 2 6.86 × 10⁵ 2.98 × 10⁻⁴ 4.35 × 10⁻¹⁰ average 5.97 ×10⁵ 2.83 × 10⁻⁴ 4.82 × 10⁻¹⁰ k_(on) k_(off) K_(D) Antibody Experiment[M⁻¹s⁻¹] [s⁻¹] [M] 4D10#3 1 6.03 × 10⁵ 3.17 × 10⁻⁴ 5.25 × 10⁻¹⁰(chimeric heavy chain; light 2 5.22 × 10⁵ 3.49 × 10⁻⁴ 6.69 × 10⁻¹⁰ chainas 4D10hum#1 and #2) average 5.63 × 10⁵ 3.33 × 10⁻⁴ 5.97 × 10⁻¹⁰ 4D10#41 4.62 × 10⁵ 2.94 × 10⁻⁴ 6.35 × 10⁻¹⁰ (heavy chain as 4D10hum#1; 2 5.06× 10⁵ 3.32 × 10⁻⁴ 6.57 × 10⁻¹⁰ chimeric light chain) average 4.84 × 10⁵3.13 × 10⁻⁴ 6.46 × 10⁻¹⁰ 4D10#5 1 4.94 × 10⁵ 2.62 × 10⁻⁴ 5.30 × 10⁻¹⁰(heavy chain as 4D10hum#2; 2 4.72 × 10⁵ 2.92 × 10⁻⁴ 6.19 × 10⁻¹⁰chimeric light chain) average 4.83 × 10⁵ 2.77 × 10⁻⁴ 5.75 × 10⁻¹⁰

Example 2.6: Analysis of Antibody Selectivity Via Dot Blot

In order to characterize the selectivity of monoclonal anti Aβ(20-42)globulomer antibodies, they were tested for binding to differentAβ-forms. To this end, serial dilutions of the individual Aβ(1-42) formsranging from 100 pmol/μl to 0.00001 pmol/μl in PBS supplemented with 0.2mg/ml BSA were prepared. 1 μl of each dilution was blotted onto anitrocellulose membrane. Detection was performed by incubating with thecorresponding antibody (0.2 μg/ml) followed by immunostaining usingPeroxidase conjugated anti-human-IgG and the staining reagent BM BluePOD Substrate (Roche).

Aβ-Standards for Dot-Blot:

1. Aβ(1-42) globulomer

Aβ(1-42) globulomer was prepared as described in Example 1a (bufferexchange by dialysis).

2. Aβ(20-42) globulomer

Aβ(20-42) globulomer was prepared as described in Example 1b.

3. Aβ(1-40) monomer, 0.1% NaOH

2.5 mg Aβ(1-40) (Bachem Inc., cat. no. H-1368) was dissolved in 0.5 ml0.1% NaOH in H₂O (freshly prepared) (=5 mg/ml) and immediately shakenfor 30 sec. at room temperature to obtain a clear solution. The samplewas stored at −20° C. until use.

4. Aβ(1-42) monomer, 0.1% NaOH

2.5 mg Aβ(1-42) (Bachem Inc., cat. no. H-1368) was dissolved in 0.5 ml0.1% NaOH in H₂O (freshly prepared) (=5 mg/ml) and immediately shakenfor 30 sec. at room temperature to obtain a clear solution. The samplewas stored at −20° C. until use.

5. Aβ(1-42) fibrils

1 mg Aβ(1-42) (Bachem Inc. cat. no.: H-1368) was dissolved in 500aqueous 0.1% NH₄OH (Eppendorf tube) and stirred for 1 min at roomtemperature. 100 μl of this freshly prepared Aβ(1-42) solution wereneutralized with 300 μl 20 mM NaH₂PO₄; 140 mM NaCl, pH 7.4. The pH wasadjusted to pH 7.4 with 1% HCl. The sample was incubated for 24 h at 37°C. and centrifuged (10 min at 10000 g). The supernatant was discardedand the fibril pellet resuspended with 400 μl 20 mM NaH₂PO₄; 140 mMNaCl, pH 7.4 by vortexing for 1 min.

6. sAPPα

Supplied by Sigma (cat. no. 59564; 25 μg in 20 mM NaH₂PO₄; 140 mM NaCl;pH 7.4). The sAPPα was diluted to 0.1 mg/ml (=1 pmol/μl) with 20 mMNaH₂PO₄, 140 mM NaCl, pH 7.4, 0.2 mg/ml BSA.

7. Aβ(12-42) globulomer

Aβ(12-42) globulomer was prepared as described in Example 1c.

Materials for Dot Blot:

Serial dilution of Aβ-standards (see above 1. to 7.) in 20 mM NaH₂PO₄,140 mM NaCl, pH 7.4+0.2 mg/ml BSA to obtain concentrations of: 100pmol/μl, 10 pmol/μl, 1 pmol/μl, 0.1 pmol/μl, 0.01 pmol/μl, 0.001pmol/μl, 0.0001 pmol/μl, and 0.00001 pmol/μl.

Nitrocellulose: Trans-Blot Transfer medium, Pure Nitrocellulose Membrane(0.2 μm); BIO-RAD

Anti-human-POD: cat no: 109-035-003 (Jackson Immuno Research)

Detection reagent: BM Blue POD Substrate, precipitating, cat no:11442066001 (Roche)

Bovine serum albumin, (BSA): Cat no: 11926 (Serva)

Blocking reagent: 5% low fat milk in TBS

Buffer solutions:

-   -   TBS: 25 mM Tris/HCl buffer pH 7.5+150 mM NaCl    -   TTBS: 25 mM Tris/HCl-buffer pH 7.5+150 mM NaCl+0.05% Tween 20    -   PBS+0.2 mg/ml BSA: 20 mM NaH2PO4 buffer pH 7.4+140 mM NaCl+0.2        mg/ml BSA

Antibody solution I: 0.2 μg/ml antibody in 20 ml 1% low fat milk in TBS

Antibody: humanized monoclonal anti-Aβ antibody 4D10hum#1; 4.7 mg/ml OD280 nm; stored at −80° C.

Antibody solution II: 1:5000 dilution of anti-human-POD in 1% low fatmilk in TBS

Dot blot procedure:

-   1) 1 μl of each of the 8 concentrations of the different    Aβ-standards (obtained by serial dilution) was dotted onto the    nitrocellulose membrane in a distance of approximately 1 cm from    each other.-   2) The dots of Aβ-standards were allowed to dry on the    nitrocellulose membrane on air for at least 10 min at room    temperature (RT). (=dot blot)-   3) Blocking:    -   The dot blot was incubated with 30 ml 5% low fat milk in TBS for        1.5 h at RT.-   4) Washing:    -   The blocking solution was discarded and the dot blot was        incubated under shaking with 20 ml TTBS for 10 min at RT.-   5) Antibody solution I:    -   The washing buffer was discarded and the dot blot was incubated        with antibody solution I for 2 h at RT-   6) Washing:    -   The antibody solution I was discarded and the dot blot was        incubated under shaking with 20 ml TTBS for 10 min at RT. The        washing solution was discarded and the dot blot was incubated        under shaking with 20 ml TTBS for 10 min at RT. The washing        solution was discarded and the dot blot was incubated under        shaking with 20 ml TBS for 10 min at RT.-   7) Antibody solution II:    -   The washing buffer was discarded and the dot blot was incubated        with antibody solution II for 1 h at RT-   8) Washing:    -   The antibody solution II was discarded and the dot blot was        incubated under shaking with 20 ml TTBS for 10 min at RT. The        washing solution was discarded and the dot blot was incubated        under shaking with 20 ml TTBS for 10 min at RT. The washing        solution was discarded and the dot blot was incubated under        shaking with 20 ml TBS for 10 min at RT.-   9) Development:    -   The washing solution was discarded. The dot blot was developed        with 7.5 ml BM Blue POD Substrate for 10 min. The development        was stopped by intense washing of the dot blot with H₂O.        Quantitative evaluation was done based on a densitometric        analysis (GS800 densitometer (BioRad) and software package        Quantity one, Version 4.5.0 (BioRad)) of the dot intensity. Only        dots were evaluated that had a relative density of greater than        20% of the relative density of the last optically unambiguously        identified dot of the Aβ(20-42) globulomer. This threshold value        was determined for every dot blot independently. The calculated        value indicates the relation between recognition of Aβ(20-42)        globulomer and the respective Aβ form for the given antibody.

Dot blot analysis was performed with humanized monoclonal anti-Aβantibody 4D10hum#1. The individual Aβ forms were applied in serialdilutions and incubated with the respective antibodies for immunereaction (1=Aβ(1-42) globulomer; 2=Aβ(20-42) globulomer; 3=Aβ(1-40)monomer, 0.1% NaOH; 4=Aβ(1-42) monomer, 0.1% NaOH; 5=Aβ(1-42) fibrilpreparation; 6=sAPPα (Sigma); (first dot: 1 pmol)). Results aresummarized in Table 8.

TABLE 8 DOT BLOT QUANTIFICATION DATA ANTIBODY: ANTIGEN 4D10hum#1Aβ(1-42) globulomer >10000 Aβ(20-42) globulomer 1 Aβ(1-40) monomer in0.1% NaOH 72000 Aβ(1-42) monomer in 0.1% NaOH 72000 Aβ(1-42)fibril >10000 sAPPα >100 Aβ(12-42) globulomer 11

Example 3: Determination of Platelet Factor 4 Cross-Reaction Example3.1: Determination of Cross-Reaction with Platelet Factor 4 inCynomolgus Monkey Plasma Via Sandwich-ELISA

Reagent List:

F96 Cert. Maxisorp NUNC-Immuno Plate cat. no. 439454

Binding antibodies in experiment E1:

-   -   Humanized monoclonal anti-Aβ antibody 4D10hum#1; 2.36 mg/ml OD        280 nm; stored at −80° C.    -   Humanized monoclonal anti-Aβ antibody 4D10hum#2; 1.74 mg/ml OD        280 nm; stored at −80° C.    -   Human/mouse chimeric anti-Aβ monoclonal antibody clone h1G5 wild        type Fc-frame (chim h1G5 wt); 0.99 mg/ml OD 280 nm; stored at        −80° C. (used as a positive control)    -   Affinity purified human polyclonal antibody hIgG1 (Chemicon        (Millipore), Cat# AG502); 1.00 mg/ml OD 280 nm; stored at        −80° C. (used as a negative control)

Binding antibodies in reference experiment R1:

-   -   Anti-HPF4 monoclonal antibody; 4.2 mg/ml OD 280 nm; Abcam cat.        no. ab49735; stored at −30° C. (used as a positive control)    -   Anti-Aβ monoclonal antibody clone m1G5; 1.70 mg/ml OD 280 nm;        stored at −80° C.    -   Anti-Aβ monoclonal antibody clone m4D10; 8.60 mg/ml OD 280 nm;        stored at −80° C.    -   Monoclonal antibody clone mIgG2a; 7.89 mg/ml OD 280 nm; stored        at −80° C. (used as a negative control)

Coating buffer: 100 mM sodium hydrogen carbonate; pH 9.6

Blocking reagent for ELISA; Roche Diagnostics GmbH cat. no.: 1112589

PBST buffer: 20 mM NaH₂PO₄; 140 mM NaCl; 0.05% Tween 20; pH 7.4

PBST+0.5% BSA buffer: 20 mM NaH₂PO₄; 140 mM NaCl; 0.05% Tween 20; pH7.4+

0.5% BSA; Serva cat. no. 11926

Cynomolgus plasma: Cynomolgus EDTA plasma pool from 13 different donors;stored at −30° C.

Trypsin inhibitor: Sigma cat. no. T7902

Primary antibody: pRAb-HPF4; 0.5 mg/ml; Abcam cat. no. ab9561

Label reagent: anti-rabbit-POD conjugate; Jackson ImmunoResearch Ltd.cat. no.: 111-036-045

Staining Solution: 42 mM TMB (Roche Diagnostics GmbH cat. no.: 92817060)in DMSO;

3% H₂O₂ in water; 100 mM sodium acetate, pH 4.9

Stop solution: 2 M sulfonic acid

Method Used in Preparation of Reagents:

Binding Antibody:

The binding antibodies were diluted to 10 μg/ml in coating buffer.

Blocking Solution:

Blocking reagent was dissolved in 100 ml water to prepare the blockingstock solution and aliquots of 10 ml were stored at −20° C. 3 mlblocking stock solution was diluted with 27 ml water for each plate toblock.

Preparation of Cynomolgus (Macaca fascicularis) Plasma Stock Solution:

2 ml Cynomolgus plasma pool were centrifuged for 10 min at 10,000 g.1.58 ml of the supernatant was removed and diluted with 3.42 mlPBST+0.5% BSA buffer (=1:3.16 dilution). Then 50 μl 10 mg/ml trypsininhibitor in H₂O were added. After incubation for 10 min at roomtemperature the sample was filtrated through a 0.22 μm filter (Milliporecat. no. SLGS0250S).

Dilution series of cynomolgus plasma stock solution:

Volume of Volume cynomolgus of PBST + 0.5% Final dilution of No plasmadilution BSA buffer cynomolgus plasma 1 250 μl stock solution 0 ml 1:3.16 2 79 μl (1) 171 μl 1:10  3 79 μl (2) 171 μl  1:31.6 4 79 μl (3)171 μl 1:100 5 79 μl (4) 171 μl 1:316 6 79 μl (5) 171 μl  1:1000 7 79 μl(6) 171 μl  1:3160 8 0 μl 250 μl buffer only

Primary Antibody Solution:

The primary antibody was diluted to 1 μg/ml in PBST+0.5% BSA buffer. Thedilution factor was 1:500. The antibody solution was used immediately.

Label Reagent:

Anti-rabbit-POD conjugate lyophilizate was reconstituted in 0.5 mlwater. 500 μl glycerol was added and aliquots of 100 μl were stored at−20° C. for further use. The concentrated label reagent was diluted inPBST buffer. The dilution factor was 1:10000. The reagent was usedimmediately.

TMB Solution:

20 ml 100 mM of sodium acetate, pH 4.9, was mixed with 200 μl of the TMBstock solution and 29.5 μl 3% peroxide solution. The solution was usedimmediately.

Standard Plate Setup for Experiment E1. Dilutions of cynomolgus plasma.Note that each sample was run in duplicate.

1 2 3 4 5 6 7 8 9 10 11 12 Positive control Negative control chim h1G5wt 4D10hum#1 4D10hum#2 hIgG1 A 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.161:3.16 1:3.16 none none none none B 1:10 1:10 1:10 1:10 1:10 1:10 1:101:10 none none none none C 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.61:31.6 1:31.6 none none none none D 1:100 1:100 1:100 1:100 1:100 1:1001:100 1:100 none none none none E 1:316 1:316 1:316 1:316 1:316 1:3161:316 1:316 none none none none F 1:1000 1:1000 1:1000 1:1000 1:10001:1000 1:1000 1:1000 none none none none G 1:3160 1:3160 1:3160 1:31601:3160 1:3160 1:3160 1:3160 none none none none H 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 none none none none

Standard Plate Setup for Reference Experiment R1. Dilutions ofcynomolgus plasma. Note that each sample was run in duplicate.

1 2 3 4 5 6 7 8 9 10 11 12 Positive control Negative control anti-HPF4mAb m1G5 mAb m4D10 mIgG2a A 1:3.16 1:3.16 1:3.16 1:3.16 1:3.16 1:3.161:3.16 1:3.16 none none none none B 1:10 1:10 1:10 1:10 1:10 1:10 1:101:10 none none none none C 1:31.6 1:31.6 1:31.6 1:31.6 1:31.6 1:31.61:31.6 1:31.6 none none none none D 1:100 1:100 1:100 1:100 1:100 1:1001:100 1:100 none none none none E 1:316 1:316 1:316 1:316 1:316 1:3161:316 1:316 none none none none F 1:1000 1:1000 1:1000 1:1000 1:10001:1000 1:1000 1:1000 none none none none G 1:3160 1:3160 1:3160 1:31601:3160 1:3160 1:3160 1:3160 none none none none H 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 none none none none

Procedure used:

-   1. 100 μl binding antibody solution per well were applied and    incubated overnight at 4° C.-   2. The antibody solution was discarded and the wells were washed    three times with 250 μl PBST buffer.-   3. 265 μl blocking solution per well were added and incubated 1.5 h    at room temperature.-   4. The blocking solution was discarded and the wells were washed    three times with 250 μl PBST buffer.-   5. After preparation of the cynomolgus plasma dilution series, 100    μl per well of these dilutions were applied to the plate. The plate    was incubated 2 h at room temperature.-   6. The cynomolgus plasma dilutions were discarded and the wells were    washed three times with 250 μl PBST buffer.-   7. 100 μl of primary antibody solution per well were added and    incubated 1 h at room temperature.-   8. The primary antibody solution was discarded and the wells were    washed three times with 250 μl PBST buffer.-   9. 200 μl label solution per well were added and incubated 1 h at    room temperature.-   10. The label solution was discarded and the wells were washed three    times with 250 μl PBST buffer.-   11. 100 μl of TMB solution were added to each well.-   12. Plate colour was monitored during development (5-15 min at    ambient temperature) and the reaction was terminated by adding 50    μl/well of stop solution when an appropriate colour had developed.-   13. The absorbance was read at 450 nm.

Data Analysis:

Plasma dilution factors (X-values) were log-transformed using theequation: X=log(X). Data were plotted using the log-transformed X-valueson the X-axis expressed as dilution of plasma (1:X). The OD_(450nm)value of the respective PBST blank in row H was subtracted from thevalues of the plasma dilution series of each column in row A-G. Theresulting background corrected OD_(450nm) values were plotted on theY-axis. The dilution effect curves were calculated from these datapoints by curve fitting using a non-linear regression “four parameterlogistic equation” with a “least squares (ordinary) fit” fitting method(that equals the fitting method “sigmoidal dose-response (variableslope)”) using the Data analysis software package GraphPadPrism (Version5.03; GraphPad Software Inc.). Curve fitting was performed for the solepurpose of data visualization but not as basis for any furthercalculations i.e. the area under the curve calculation. The area underthe curve (AUC, or total peak area) was determined based on non-curvefitted data, the log-transformed X-values and the OD_(450nm) values inthe measured range (final plasma dilutions from 1:3.16 to 1:3160). Thefollowing calculation settings were used within the Data analysissoftware package GraphPadPrism (Version 5.03; GraphPad Software Inc.):

-   -   The baseline was set to Y=0.0.    -   Minimum peak height: Ignore peaks that are less than 10% of the        distance from minimum to maximum Y.    -   Peak direction: By definition, all peaks must go above the        baseline

For each individual antibody a PF4 discrimination factor was calculatedusing the commercially available anti-HPF4 antibody (Abeam cat. no.:ab49735) as a reference antibody for PF4 recognition, wherein

$\left\lbrack {{PF}\; 4\mspace{14mu}{discrimination}\mspace{14mu}{factor}} \right\rbrack = \frac{\left\lbrack {{total}\mspace{14mu}{peak}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{anti}\text{-}{HPF}\; 4\mspace{14mu}{antibody}\mspace{14mu}{ab}\; 49735} \right\rbrack}{\left\lbrack {{total}\mspace{14mu}{peak}\mspace{14mu}{area}\mspace{14mu}{of}\mspace{14mu}{antibody}\mspace{14mu}{to}\mspace{14mu}{be}\mspace{14mu}{determined}} \right\rbrack}$

Note: The PF4 discrimination factor was calculated based on theanti-HPF4 antibody AUCs obtained in the reference experiment as no humanversion of an anti-HPF4 exists.

Results of experiment E1 and reference experiment R1 are shown in FIGS.20A and 22A as well as in Tables 9A and 9B.

Example 3.2: Determination of Cross-Reaction with Platelet Factor 4 inHuman Plasma Via Sandwich-ELISA

The same reagents and procedures for reagent preparation were used asfor Example 3.1 except from:

Human plasma (Human EDTA plasma pool from 4 different donors; stored at−30° C.) spiked with human PF4 (7.3 mg/ml; Molecular Innovation cat. no.HPF4; stored at −30° C.) was used instead of cynomolgus plasma.HPF4-spiked human plasma stock solution was prepared as follows.

A) Preparation of Human Plasma Dilution:

2 ml human plasma pool were centrifuged for 10 min at 10000 g. 1.58 mlof the supernatant was removed and diluted with 3.42 ml PBST+0.5% BSA(=1:3.16 dilution). Then 50 μl 10 mg/ml trypsin inhibitor in H₂O wereadded. After incubation for 10 min at room temperature the sample wasfiltrated through a 0.22 μm filter (Millipore cat. no. SLGS0250S).

B) Preparation of HPF4 Stock Solution:

1 μl HPF4 was added to 99 μl PBST+0.5% BSA buffer=73 μg/ml.

C) Preparation of Human Plasma Stock Solution Spiked with 10 ng/ml HPF4:

0.69 μl of 73 μg/ml HPF4 stock solution were added to 5 ml 1:3.16diluted human plasma resulting in 10 ng/ml HPF4 spiking of the humanplasma stock dilution.

The preparation of a dilution series, the standard plate setup, theexperimental procedure and the data analysis for sandwich-ELISA withHPF4-spiked human plasma were analogous to those described forsandwich-ELISA with cynomolgus plasma in Example 3.1.

Binding antibodies in experiment E2: same as used in experiment E1 inExample 3.1 Binding antibodies in reference experiment R2: same as usedin reference experiment R1 in Example 3.1.

Results of experiment E2 and reference experiment R2 are shown in FIGS.20B and 22B as well as in Tables 9A and 9B.

TABLE 9A AUC (OR TOTAL PEAK AREA) CALCULATED FROM LOG- TRANSFORMED DATAOF EXPERIMENTS E1 and E2 DEPICTED IN FIGS. 20A AND 20B Positive Negativecontrol mAb mAb control chim h1G5 wt¹ 4D10hum#1 4D10hum#2 hIgG1Cynomolgus Area Under Curve² 1.255 0.042 0.075 0.075 plasma Ratio 2 6436 27 (data from HPF4/aAβ FIG. 20A) antibody Human plasma Area UnderCurve² 0.949 0.067 0.116 0.113 (data from Ratio 2 30 17 18 FIG. 20B)HPF4/aAβ antibody ¹chim h1G5 wt is an antibody as described in WO2007/062853, i.e. a monoclonal antibody having a binding affinity to theAβ(20-42) globulomer that is greater than its binding affinity to theAβ(1-42) globulomer. ²Area under curve was calculated as described inexample 3.1.

TABLE 9B AUC (OR TOTAL PEAK AREA) CALCULATED FROM LOG- TRANSFORMED DATAOF REFERENCE EXPERIMENTS R1 and R2 DEPICTED IN FIGS. 22A AND 22BPositive Negative control mAb mAb control anti-HPF4 m1G5¹ m4D10 mIgG2aCynomolgus plasma Area Under Curve² 2.681 0.861 0.086 0.005 (data fromFIG. 22A) Ratio 1 3 31 517 HPF4/aAβ antibody Human plasma Area UnderCurve² 1.986 0.311 0.093 0.006 (data from FIG. 22B) Ratio 1 6 21 331HPF4/aAβ antibody ¹m1G5 is an antibody as described in WO 2007/062853,i.e. a monoclonal antibody having a binding affinity to the Aβ(20-42)globulomer that is greater than its binding affinity the Aβ(1-42)globulomer. ²Area under curve was calculated as described in example3.1.

Example 3.3: Determination of Cross-Reaction with Platelet Factor 4 inCynomolus Monkey Plasma Via Aligned Sandwich-ELISA

The reagents described in Example 3.1 and aligning antibodies anti-mouseIgG (Fc specific; produced in goat; Sigma cat. no.: M3534; 2.3 mg/ml;stored at −20° C. for murine binding antibodies in reference experimentR3) and anti-human IgG (Fc specific; produced in goat; Sigma cat. no.:12136; 2.2 mg/ml; stored at −20° C., for human, humanized andhuman/mouse chimeric binding antibodies in experiment E3) were used.

Methods Used in Preparation of Reagents:

Blocking solution, primary antibody and TMB solution were prepared asdescribed in Example 3.1.

Each aligning antibody was diluted to 10 μg/ml in coating buffer.

Binding antibodies in experiment E3: same as used in experiment E1 inExample 3.1 Binding antibodies in reference experiment R3: same as usedin reference experiment R1 in Example 3.1.

Each binding antibody was diluted with PBST+0.5% BSA buffer to 10 ng/ml(stock solution), and dilution series were prepared as follows:

Volume Volume of of PBST + 0.5% Final antibody No antibody dilution BSAbuffer concentration 1 250 μl stock solution 0 ml 10000 ng/ml  2 79 μl(1) 171 μl 3160 ng/ml 3 79 μl (2) 171 μl 1000 ng/ml 4 79 μl (3) 171 μl 316 ng/ml 5 79 μl (4) 171 μl  100 ng/ml 6 79 μl (5) 171 μl  31.6 ng/ml7 79 μl (6) 171 μl  10 ng/ml 8 0 μl 250 μl buffer only

Cynomolgus Plasma:

400 μl Cynomolgus plasma pool were centrifuged for 10 min at 10000 g.158 of the supernatant was removed and diluted with 684 μl PBST+0.5% BSA(=1:3.16 dilution). Then 10 μl 10 mg/ml trypsin inhibitor in H₂O wereadded. After incubation for 10 min at room temperature the sample wasfiltrated through a 0.22 μm filter (Millipore cat. no. SLGS0250S).Afterwards 500 of this 1:3.16 diluted plasma sample was again diluted1:31.6 with 15.3 ml PBST+0.5% BSA buffer resulting in a total dilutionof 1:100.

Label Reagent:

Anti-rabbit-POD conjugate lyophilised was reconstituted in 0.5 ml water.500 μl glycerol was added and aliquots of 100 μl were stored at −20° C.for further use. The concentrated label reagent was diluted in PBSTbuffer. The dilution factor was 1:5000. The reagent was usedimmediately.

Binding Antibody Plate Setup for Experiment E2. Dilutions of bindingantibodies. Note that each concentration of each binding antibody wasrun in duplicate.

1 2 3 4 5 6 7 8 9 10 11 12 Positive control Negative control chim h1G5wt 4D10hum#1 4D10hum#2 hIgG1 A 10000 10000 10000 10000 10000 10000 1000010000 none none none none B 3160 3160 3160 3160 3160 3160 3160 3160 nonenone none none C 1000 1000 1000 1000 1000 1000 1000 1000 none none nonenone D 316 316 316 316 316 316 316 316 none none none none E 100 100 100100 100 100 100 100 none none none none F 31.6 31.6 31.6 31.6 31.6 31.631.6 31.6 none none none none G 10 10 10 10 10 10 10 10 none none nonenone H 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 none none none none

Binding Antibody Plate Setup for Reference Experiment R3. Dilutions ofbinding antibodies. Note that each concentration of each bindingantibody was run in duplicate.

1 2 3 4 5 6 7 8 9 10 11 12 Positive control Negative control anti-HPF4mAb m1G5 mAb m4D10 mIgG2a A 10000 10000 10000 10000 10000 10000 1000010000 none none none none B 3160 3160 3160 3160 3160 3160 3160 3160 nonenone none none C 1000 1000 1000 1000 1000 1000 1000 1000 none none nonenone D 316 316 316 316 316 316 316 316 none none none none E 100 100 100100 100 100 100 100 none none none none F 31.6 31.6 31.6 31.6 31.6 31.631.6 31.6 none none none none G 10 10 10 10 10 10 10 10 none none nonenone H 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 none none none none

Procedure used:

-   1. 100 μl of the respective aligning antibody solution (anti-human    IgG for experiment E3; anti-murine IgG for reference experiment R3)    per well were applied and incubated overnight at 4° C.-   2. The antibody solution was discarded and the wells were washed    three times with 250 μl PB ST-buffer.-   3. 265 μl blocking solution per well were added and incubated 2 h at    room temperature.-   4. The blocking solution was discarded and the wells were washed    three times with 250 μl PBST buffer.-   5. After preparation of the dilution series of each binding    antibody, 100 μl per well of these antibody dilutions were applied    to the plate. The plate was incubated 2 h at room temperature.-   6. The antibody solutions were discarded and the wells were washed    three times with 250 μl PBST buffer.-   7. 100 μl 1:100 dilution of cynomolgus plasma per well were added    and incubated 2 h at room temperature.-   8. The plasma solution was discarded and the wells were washed three    times with 250 μl PBST buffer.-   9. 100 μl primary antibody solution per well were added and    incubated 1 h at room temperature.-   10. The primary antibody solution was discarded and the wells were    washed three times with 250 μl PBST buffer.-   11. 200 μl label reagent per well were added and incubated 1 h at    room temperature.-   12. The label reagent was discarded and the wells were washed three    times with 250 μl PBST buffer.-   13. 100 μl of TMB solution were added to each well.-   14. Plate colour was monitored during development (5-15 min at    ambient temperature) and the reaction was terminated by adding 50    μl/well of stop solution when an appropriate colour had developed.-   15. The absorbance was read at 450 nm.

Data analysis was performed as described for sandwich-ELISA withcynomolgus plasma in Example 3.1, except that not plasma dilutionfactors but the amounts of antibody (expressed in ng/ml) were used asX-values and thus concentration effect curves were calculated.Accordingly, area under curve was determined based on non-curve fitteddata, the log-transformed X-values and the OD_(450nm) values in themeasured range (final antibody concentrations from 10 ng/ml to 10000ng/ml).

Results of experiment E3 and reference experiment R3 are shown in FIGS.21A and 23A as well as in Tables 10A and 10B.

Example 3.4: Determination of Cross-Reaction with Platelet Factor 4 inHuman Plasma Via Aligned Sandwich-ELISA

The same reagents and procedures for reagent preparation were used asfor Example 3.3 except from:

Each aligning antibody used for experiment E4 was diluted to 10 μg/ml incoating buffer, and each aligning antibody used for experiment R4 wasdiluted to 50 μg/ml in coating

Human plasma (Human EDTA plasma pool from 4 different donors; stored at−30° C.) spiked with human PF4 (7.3 mg/ml; Molecular Innovation cat. no.HPF4; stored at −30° C.) was used instead of cynomolgus plasma.HPF4-spiked human plasma stock solution was prepared as follows.

A) Preparation of Human Plasma Dilution:

4 ml human plasma pool were centrifuged for 10 min at 10000 g. 3.16 mlof the supernatant was removed and diluted with 6.84 ml PBST+0.5% BSA(=1:3.16 dilution). Then 100 μl 10 mg/ml trypsin inhibitor in H₂O wereadded. After incubation for 10 min at room temperature the sample wasfiltrated through a 0.22 μm filter (Millipore cat. no. SLGS0250S).Afterwards 5 ml of this 1:3.16 diluted plasma sample was again diluted1:3.16 with 10.8 ml PBST+0.5% BSA buffer resulting in a total dilutionof 1:10.

B) Preparation of HPF4 Stock Solution:

1 μl HPF4 was added to 99 μl PBST+0.5% BSA buffer=73 μg/ml.

C) Preparation of Human Plasma Stock Solution Spiked with 10 ng/ml HPF4:

1.64 of 73 μg/ml HPF4 stock solution were added to 12 ml 1:10 dilutedhuman plasma resulting in 10 ng/ml HPF4 spiking of the human plasmastock dilution.

The preparation of dilution series of the binding antibodies; thebinding antibody plate setup; the preparation of blocking solution,primary antibody, reagent and TMB solution were the same as in Example3.3.

Aligning antibody and binding antibodies in experiment E4: same as usedin experiment E3 in Example 3.3

Aligning antibody and binding antibodies in reference experiment R4:same as used in reference experiment R4 in Example 3.3.

The experimental procedure (but using 1:10 diluted human plasma in step7) and data analysis for aligned sandwich-ELISA with HPF4-spiked humanplasma were analogous to that described for aligned sandwich-ELISA withcynomolgus plasma in Example 3.3.

Results of experiment E4 and reference experiment R4 are shown in FIGS.21B and 23B as well as in Tables 10A and 10B.

TABLE 10A AUC (OR TOTAL PEAK AREA) CALCULATED FROM LOG- TRANSFORMED DATAOF EXPERIMENTS E3 and E4 DEPICTED IN FIGS. 21A AND 21B Positive mAb mAbNegative control 4D10 4D10 control chim h1G5 wt¹ hum#1 hum#2 hIgG1Cynomolgus plasma Area Under Curve² 0.290 0.030   0   0 (data from FIG.21A) Ratio 16 158 >158³ >158³ HPF4/aAβ antibody Human plasma Area UnderCurve² 0.106 0.168   0.051   0.024 (data from FIG. 21B) Ratio 36 23  75 157 HPF4/aAβ antibody ¹chim h1G5 wt is an antibody as described in WO2007/062853, i.e. a monoclonal antibody having a binding affinity to theAβ(20-42) globulomer that is greater than its binding affinity to theAβ(1-42) globulomer. ²Area under curve was calculated as described inexample 3.3. ³For antibodies 4D10hum#2 and hIgG1 the HPF4 bindingactivity was so low that the AUC was calculated to be 0. Therefore theratio HPF4/aAβ antibody could not be calculated and was indicated tobe >158 (the highest ratio achieved by another antibody (4D10hum#1) inthis assay).

TABLE 10B AUC (OR TOTAL PEAK AREA) CALCULATED FROM LOG- TRANSFORMED DATAOF REFERENCE EXPERIMENTS R3 and R4 DEPICTED IN FIGS. 21A AND 21BPositive Negative control mAb mAb control anti-HPF4 m1G5¹ m4D10 mIgG2aCynomolgus plasma Area Under Curve² 4.781 0.2768 0.04066 0.01473 (datafrom FIG. 23A) Ratio 1 17 118 325 HPF4/aAβ antibody Human plasma AreaUnder Curve² 3.844 0.165 0.141 0.033 (data from FIG. 23B) Ratio 1 23 27118 HPF4/aAβ antibody ¹m1G5 is an antibody as described in WO2007/062853, i.e. a monoclonal antibody having a binding affinity to theAβ(20-42) globulomer that is greater than its binding affinity to boththe Aβ(1-42) globulomer. ²Area under curve was calculated as describedin example 3.3.

What is claimed is:
 1. A method for treating a subject for Alzheimer'sdisease by administering to the subject an effective amount of ananti-Aβ(20-42) globulomer antibody comprising: a first amino acidsequence which is at least 90% identical to SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, or SEQ ID NO:11; and a second amino acid sequencewhich is at least 90% identical to SEQ ID NO:1, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16, wherein the firstamino acid sequence comprises three complementarity determining regionsconsisting of amino acids 31-35, 50-65, and 98-101, respectively, of SEQID NO:2 or SEQ ID NO:3; and wherein the second amino acid sequencecomprises three complementarity determining regions consisting of aminoacids 24-39, 55-61, and 94-102, respectively, of SEQ ID NO:1.
 2. Themethod of claim 1, wherein the first amino acid sequence is at least 90%identical to SEQ ID NO:2 or SEQ ID NO:3; and a second amino acidsequence is at least 90% identical to SEQ ID NO:1.
 3. The method ofclaim 1, wherein the first amino acid sequence is at least 90% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:11.
 4. The method of claim 1, wherein thesecond amino acid sequence is at least 90% identical to an amino acidsequence selected from the group consisting of SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.
 5. The method ofclaim 1, wherein the first amino acid sequence is at least 90% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:11; and the second amino acid sequence is atleast 90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,and SEQ ID NO:16.
 6. The method of claim 1, wherein the antibody isselected from the group consisting of: an immunoglobulin molecule, amonoclonal antibody, a chimeric antibody, a CDR-grafted antibody, ahumanized antibody, a multispecific antibody, a dual variable domain,and a bispecific antibody.
 7. The method of claim 1, wherein theantibody further comprises an immunoglobulin light chain constant regionhaving an amino acid sequence selected from the group consisting of SEQID NO:27 and SEQ ID NO:28.
 8. The method of claim 1, wherein theantibody further comprises an agent selected from the group consistingof: an immunoadhesion molecule; an imaging agent, and a therapeuticagent.
 9. The method of claim 1, wherein the antibody possesses a humanglycosylation pattern.
 10. A method for treating a subject forAlzheimer's disease by administering to the subject an effective amountof a pharmaceutical composition comprising: an anti-Aβ(20.42) globulomerantibody comprising: a first amino acid sequence which is at least 90%identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ IDNO:11; and a second amino acid sequence which is at least 90% identicalto SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,or SEQ ID NO:16, wherein the first amino acid sequence comprises threecomplementarity determining regions consisting of amino acids 31-35,50-65, and 98-101, respectively, of SEQ ID NO:2 or SEQ ID NO:3; andwherein the second amino acid sequence comprises three complementaritydetermining regions consisting of amino acids 24-39, 55-61, and 94-102,respectively, of SEQ ID NO:1; and a pharmaceutically acceptable carrier.11. The method of claim 10, wherein the pharmaceutical compositionfurther comprises at least one additional therapeutic agent.
 12. Themethod of claim 1, wherein the antibody has a binding affinity to anAβ(20-42) globulomer that is greater than the binding affinity of theantibody to an Aβ(1-42) globulomer.
 13. The method of claim 10, whereinthe antibody has a binding affinity to an Aβ(20-42) globulomer that isgreater than the binding affinity of the antibody to an Aβ(1-42)globulomer.
 14. The method of claim 1, wherein the first amino acidsequence is selected from the group consisting of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,and SEQ ID NO:11; and the second amino acid sequence is selected fromthe group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, and SEQ ID NO:16.
 15. The method of claim 10, wherein the firstamino acid sequence is selected from the group consisting of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:11; and the second amino acid sequence isselected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, and SEQ ID NO:16.